Methods and systems for adenovirus interaction with desmoglein 2 (DSG2)

ABSTRACT

The present invention provides compositions and methods for treating disorders associated with epithelial tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/353,652, filed Jun. 10, 2010, U.S. Provisional Application No.61/430,091, filed Jan. 5, 2011, and U.S. Provisional Application No.61/470,663, filed Apr. 1, 2011, all of which are incorporated herein byreference in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with U.S. Government support under R01 CA080192and RO1 HLA078836 awarded by the National Institutes of Health. The U.S.Government has certain rights in this invention.

BACKGROUND

Human adenoviruses (Ads) have been classified into six species (A to F),currently containing 51 serotypes. Most Ad serotypes utilize thecoxsackie-adenovirus receptor (CAR) as a primary attachment receptor(Bergelson et al., 1997). This is, however, not the case for species BAd serotypes. Recently, we have suggested a new grouping of species BAds based on their receptor usage (Tuve et al., 2006). Group 1 (Ad16,21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3,Ad7, 14) share a common, unidentified receptor/s, which is not CD46 andwhich was tentatively named receptor X; Group 3 (Ad11) preferentiallyinteracts with CD46, but also utilizes receptor X if CD46 is blocked.

Species B Ads are common human pathogens. Since 2005, a simultaneousemergence of diverse species B serotypes at the majority of US militarytraining facilities was observed. This included serotypes Ad3, Ad7, andAd14 (Metzgar et al., 2007). In 2007 a new, highly pathogenic strain andpossibly more virulent strain of Ad14, Ad14a, has been discovered atseveral sites in the US and in Asia (Louie et al., 2008; Tate et al.,2009). We recently demonstrated that Ad14a belongs to species B group 2Ads with regards to their receptor usage (Wang et al., 2009).Collectively, all receptor X utilizing serotypes (Ad3, Ad7, Ad14, Ad14a,and Ad11) are referred to herein as AdB-2/3.

AdB-2/3 have great relevance as gene transfer vectors, particularly withregard to tumors of epithelial origin, representing most solid tumors(Yamamoto and Curiel, 2010). Epithelial cells maintain severalintercellular junctions and an apical-basal polarity. Key features ofepithelial cells are conserved in epithelial cancers in situ and incancer cell lines (Turley et al., 2008). Both CAR and CD46 are oftentrapped in tight and adherence junctions of epithelial cancer cells andare not accessible to Ads that use these attachment receptors (Coyne andBergelson, 2005; Strauss et al., 2009). In contrast, AdB-2/3 efficientlyinfect epithelial cancer cells, which is accomplished in part throughinduction of processes that are reminiscent of Epithelial-to-MesenchymalTransition (EMT) (Strauss et al., 2009). Another distinctive feature ofAdB-2/3 is their ability to produce subviral dodecahedral particlesduring their replication, consisting of Ad fiber and penton base (Norrbyet al., 1967). Penton-Dodecahedra (PtDd) cannot assemble fromfull-length penton base protein, but require spontaneous N-terminaltruncation by proteolysis between residues 37 and 38 (Fuschiotti et al.,2006). This cleaved site is conserved in Ad3, Ad7, Ad11, and Ad14 but isnot present in Ad2 and Ad5. In the case of Ad3 the PtDd are formed at amassive excess of 5.5×10⁶ PtDd per infectious virus (Fender et al.,2005), and it has been suggested that PtDd enhance Ad3 infectivity bydisturbing intercellular junctions, thus favoring virus spreading(Walters et al., 2002).

The first attempts to identify receptor X date back to 1995. Recently,several candidates for receptor X such as CD46, CD80 and/or CD86 weresuggested (Fleischli et al., 2007; Short et al., 2004; Short et al.,2006; Sirena et al., 2004). However, no one thus far has been able toverify that these proteins can serve as the high affinity receptor forAdB-2/3 (Gaggar et al., 2003b; Gustafsson et al., 2006; Marttila et al.,2005; Persson et al., 2008; Segerman et al., 2003; Tuve et al., 2006).

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods for enhancingtherapeutic treatment, or diagnosis of a disorder associated withepithelial tissue, and/or imaging epithelial tissues, comprisingadministering to a subject in need thereof:

-   -   a) an amount of one or more therapeutic sufficient to treat the        disorder, diagnostic sufficient to diagnose the disorder, and/or        imaging agent sufficient to image the epithelial tissue; and    -   b) an amount of AdB-2/3 fiber multimer or functional equivalent        thereof sufficient to enhance efficacy of the one or more        therapeutics, diagnostics, and/or imaging agents.

The method of the invention can be used to treat and/or diagnosedisorders associated with epithelial tissue, and/or image epithelialtissues, such as epithelial tissues in which such a disorder is believedpresent, where such disorders include but are not limited to solidtumors, irritable bowel syndrome, inflammatory bowel disorder, Crohn'sdisease, ulcerative colitis, constipation, gatroesophageal refluxdisease, chronic obstructive pulmonary disease, asthma, bronchitis,pulmonary emphysema, cystic fibrosis, interstitial lung disease,pneumonia, primary pulmonary hypertension, pulmonary embolism, pulmonarysarcoidosis, tuberculosis, pancreatitis, pancreatic duct disorders,brain disorders (ie: any brain disorder that could benefit from improvedtransport of drugs through the epithelial blood-brain barrier), bileduct obstruction, cholecystitis, choledocholithiasis, infections(including but not limited to cellulitis, pneumonia, andpyelonephritis), and gallstones. In a preferred embodiment, the disorderis a solid tumor, including, but not limited to, breast tumors, lungtumors, colon tumors, rectal tumors, stomach tumors, prostate tumors,ovarian tumors, uterine tumors, cervical tumors, kidney tumors, skintumors, melanomas, pancreatic tumors, liver tumors, endocrine tumors,brain tumors, head and neck tumors, nasopharyngeal tumors, gastrictumors, squamous cell carcinomas, adenocarcinomas, bladder tumors, andesophageal tumors.

The methods of any embodiment of the invention can utilize, for example,AdB-2/3 fiber multimers selected from the group consisting of an Ad3fiber multimer, and Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14fiber multimer, an Ad14a fiber multimer, combinations thereof, andfunctional equivalents thereof. In a preferred embodiment, the AdB-2/3fiber multimer is an Ad3 fiber multimer, or functional equivalentsthereof. In further embodiments, the AdB-2/3 fiber multimer can compriseAdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd),recombinant AdB-2/3 fiber multimers, and functional equivalents thereof.In preferred embodiments, the AdB-2/3 fiber multimer comprises an Ad3PtDd or junction opener 1 (JO-1) (SEQ ID NO:20)

Any suitable therapeutic, diagnostic, or imaging agent can be used inthese methods. In embodiments where therapeutics are used, suchtherapeutics may include but are not limited to antibodies,immunoconjugates, vaccines, radioactive particle/radiation therapy(“radiation”), chemotherapeutics, cellular immunotherapy includingadoptive T-cell therapy and dendritic cell therapy, nanoparticles,inhaled therapeutics, gene therapy constructs, and nucleic acidtherapeutics or combinations thereof. In preferred embodiments, thetherapeutic comprises a chemotherapeutic or an anti-tumor monoclonalantibody. Exemplary anti-tumor monoclonal antibodies that can be used inthe methods of the invention include, but are not limited totrastuzumab, cetumiximab, petuzumab, apomab, conatumumab, lexatumumab,bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab,edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab,epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomabdaclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.

In one preferred embodiment, the disorder associated with epithelialtissue comprises a Her-2 positive tumor. Exemplary Her-2 positive tumorsthat can be treated under this embodiment include, but are not limitedto, breast tumors, gastric tumors, colon tumors, and ovarian tumors. Inthis embodiment, it is further preferred that the therapeutic comprisestrastuzumab; in a further preferred embodiment, the therapeutic furthercomprises one or more chemotherapeutics and/or radiation. In thisembodiment, it is further preferred that the AdB-2/3 fiber multimercomprises an Ad3 PtDd, JO-1 (SEQ ID NO:20), or functional equivalentsthereof. In another preferred embodiment, the subject to be treated hasnot responded to trastuzumab therapy.

In another preferred embodiment, the disorder associated with epithelialtissue comprises an EGFR-positive tumor. Exemplary EGFR-positive tumorsinclude, but are not limited to lung tumors, colon tumors, breasttumors, rectal tumors, head and neck tumors, and pancreatic tumors. Inthis embodiment, it is preferred that the therapeutic comprisescetuximab; in a further preferred embodiment, the therapeutic furthercomprises one or more chemotherapeutics and/or radiation. In thisembodiment, it is further preferred that the AdB-2/3 fiber multimercomprises an Ad3 PtDd, JO-1 (SEQ ID NO:20), or functional equivalentsthereof. In another preferred embodiment, the subject to be treated hasnot responded to cetuximab therapy.

In another embodiment, the disorder associated with epithelial tissuecomprises a solid tumor and therapeutic comprises a vascular endothelialgrowth factor (VEGF) inhibitor.

In a second aspect, the present invention provides methods for treatinga disorder associated with epithelial tissue, comprising administeringto a subject in need thereof an amount of AdB-2/3 fiber multimer orfunctional equivalent thereof, sufficient to treat the disorder. In thisembodiment, the AdB-2/3 fiber multimer is administered as a monotherapy.In one embodiment, the method is used to treat an AdB-2/3 viralinfection. In another embodiment, the disorder to be treated is a solidtumor. Exemplary solid tumors that can be treated using this aspect ofthe invention include but are not limited to breast tumors, lung tumors,colon tumors, rectal tumors, stomach tumors, prostate tumors, ovariantumors, uterine tumors, cervical tumors, kidney tumors, skin cancers,melanomas, pancreatic tumors, liver tumors, endocrine tumors, braintumors, head and neck tumors, nasopharyngeal tumors, gastric tumors,squamous cell carcinomas, adenocarcinomas, bladder tumors, andesophageal tumors. In this aspect, exemplary AdB-2/3 fiber multimers foruse include, but are not limited to an Ad3 fiber multimer, and Ad7 fibermultimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fibermultimer, combinations thereof, and functional equivalents thereof. In apreferred embodiment, the AdB-2/3 fiber multimer is an Ad3 fibermultimer, and functional equivalents thereof. In a further preferredembodiment, the AdB-2/3 fiber multimer is selected from the groupconsisting of AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedralparticles (PtDd), recombinant AdB-2/3 fiber multimers, and functionalequivalents thereof. In various further preferred embodiments, theAdB-2/3 fiber multimer comprises an Ad3 PtDd or junction opener 1 (JO-1)(SEQ ID NO:20),

In a third aspect, the present invention provides recombinant AdB-2/3fiber polypeptides, comprising:

-   -   a) one or more AdB-2/3 fiber polypeptide shaft domains, or        functional equivalents thereof;    -   b) an AdB-2/3 fiber polypeptide knob domain, or functional        equivalent thereof. operatively linked to and located C-terminal        to the one or more AdB-2/3 fiber polypeptide shaft domains; and    -   c) one or more non-AdB-2/3-derived dimerization domains        operatively linked to and located N-terminal to the one or more        AdB-2/3 fiber polypeptide shaft domains.

In one embodiment, the recombinant AdB-2/3 fiber polypeptide does notinclude an AdB-2/3 tail domain. In another embodiment, each shaft domainis selected from the group consisting of an Ad3 shaft domain, an Ad7shaft domain, an Ad11 shaft domain, an Ad 14 shaft domain, an Ad14ashaft domain, combinations thereof, and functional equivalents thereof.In a further embodiment, the one or more shaft domains comprise 1-22shaft domains. In various further embodiments, each shaft domaincomprises or consists of an amino acid sequence according to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11or SEQ ID NO:12. In a further embodiment, the knob domain is selectedfrom the group consisting of an Ad3 knob domain, an Ad7 knob domain, anAd11 knob domain, an Ad 14 knob domain, an Ad14a knob domain, andfunctional equivalents thereof. In various further embodiments, the knobdomain comprises or consists of an amino acid sequence according to SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:13 or SEQ ID NO:14. In a further embodiment, the dimerization domaincomprises an amino acid sequence selected from the group consisting ofEVSALEK (SEQ ID NO: 22) and/or KVSALKE (SEQ ID NO: 23). In a preferredembodiment, the recombinant AdB-2/3 polypeptide comprises: (a) one ormore shaft domains that each comprise or consist of an Ad3 shaft domain(SEQ ID NO: 1); and (b) a knob domain comprises or consists of an Ad3knob domain (SEQ ID NO:6).

In a further preferred embodiment, the recombinant AdB-2/3 fiberpolypeptide comprises or consists of the amino acid sequence of JO-1(SEQ ID NO:20), In a further preferred embodiment, the AdB-2/3 fiberpolypeptide is multimerized; most preferably dimerized.

In other aspects, the present invention provides isolated nucleic acidsencoding the recombinant AdB-2/3 fiber polypeptides of the invention;recombinant expression vectors comprising the isolated nucleic acids ofthe invention; host cells comprising the recombinant expression vectorsof the invention; and pharmaceutical compositions comprising AdB-2/3multimers as described herein. In a further aspect, the presentinvention provides methods for improving delivery of a compound to anepithelial tissue, comprising contacting the epithelial tissue with (a)one or more compound to be delivered to the epithelial tissue; and (b)an amount of AdB-2/3 fiber multimer, or functional equivalent thereof,sufficient to enhance delivery of the one or more compounds to theepithelial tissue. The methods permit improved delivery of any compoundthat targets epithelial cells, including but not limited to diagnosticcompounds and imaging compounds. In one preferred embodiment, theepithelial tissue comprises a solid tumor.

In a still further aspect, the present invention provides methods forimproving delivery of a compound to tissue expressing desmoglein 2(DSG2), comprising contacting the tissue expressing DSG2 with (a) one ormore compound to be delivered to the tissue; and (b) an amount ofAdB-2/3 fiber multimer, or functional equivalent thereof, sufficient toenhance delivery of the one or more compounds to the tissue. The methodsof this aspect of the invention can be used to improve delivery of anycompound of interest to a tissue expressing DSG2.

In a yet further aspect, the present invention provides methods forinducing an epithelial to mesenchymal transition (EMT) in a tissue,comprising contacting the epithelial tissue with an amount of AdB-2/3fiber multimer, or functional equivalent thereof, sufficient to induceEMT.

In one preferred embodiment of each of these further aspects of theinvention, the AdB-2/3 fiber multimer is selected from the groupconsisting of an Ad3 fiber multimer, and Ad7 fiber multimer, an Ad11fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer,combinations thereof, and functional equivalents thereof. In variousfurther preferred embodiments, the AdB-2/3 fiber multimer is an Ad3fiber multimer, or a functional equivalent thereof; the AdB-2/3 fibermultimer is selected from the group consisting of AdB-2/3 virions,AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd), recombinantAdB-2/3 fiber multimers, and functional equivalents thereof; the AdB-2/3fiber multimer comprises an Ad3 PtDd; the AdB-2/3 fiber multimercomprises junction opener 1 (JO-1) (SEQ ID NO:20); and the AdB-2/3 fibermultimer is a dimer.

In another aspect, the present invention provides method for identifyingcandidate compounds for one or more of treating a disorder associatedwith epithelial tissue, improving delivery of a substance to anepithelial tissue, for improving delivery of a substance tissueexpressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3infection comprising (a) contacting an AdB-2/3 fiber multimer to DSG2under conditions to promote multimer binding to DSG2, wherein thecontacting is carried out in the presence of one or more test compounds;and (b) identifying positive test compounds that compete for binding ofthe AdB-2/3 fiber multimer to DSG2 compared to control; wherein thepositive test compounds are candidate compounds for one or more oftreating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, for improving deliveryof a substance tissue expressing DSG2, inducing an EMT in a tissue,and/or treating an AdB-2/3 infection.

DESCRIPTION OF THE FIGURES

FIG. 1. Tools for Ad receptor identification and competition studies forAd attachment.

a) Amino acid alignment of N-termini of Ad pentons. The proteasecleavage site at aa 37/38 is marked in orange. b) Scheme of viralparticles and particle components used in this study. PtDd possess 12units containing penton base and trimeric fiber. BsDd only containpenton base. c) Competition of ³H-labeled Ad14, Ad14a, Ad11 and Ad35virus attachment to HeLa cells after pre-incubation with Ad3 BsDd, PtDd,or antiCD46 antibodies (aCD46). Attachment in PBS-treated cells wastaken as 100%. n=5. Notably, the finding that PtDd partially blocks Ad35could be due to the physical proximity of DSG2 and CD46 in HeLa cells.d) Ad3 attachment studies as in c) after preincubation with both Ad3fiber knob and BsDd (BsDd+Ad3K). The molar concentration of Ad3K wasequal that of fiber knob within PtDd used for competition. e) Scheme ofAd3-GFP vector. The vector is based on wild-type Ad deleted for nt29,892-30,947 to accommodate the CMV-GFP-polyA expression cassette. Thelower panel shows restriction enzyme analyses of wt Ad3 and Ad3-GFP withthe expected fragments. M: DNA size marker. f and g) Validation ofpolyclonal rabbit antibody against recombinant Ad3 fiber knob. f)Western blot. Ad knobs (Ad3K, Ad5K, 14K, 14aK, Ad11K, Ad35K) or PtDdwere separated in PAG cells with (B) and without (UB) sampledenaturation. Filters were incubated with antiAd3K antiserum andanti-rabbit-HRP antibodies. g) Inhibition of Ad attachment: ³H-labeledAd3, Ad14 and Ad5 virus were incubated with PBS (white bars) or rabbitanti-Ad3K serum (grey bars) for one hour on ice, then added to HeLacells for attachment studies.

FIG. 2. Identification of receptor X using Ad3 virions and Ad3 PtDd.

a) Competition of ³H-labeled Ad3 and Ad5 virus attachment to HeLa cellsafter pre-incubation with Ad3 BsDd, PtDd, or Ad fiber knobs. Attachmentin PBS-treated cells was taken as 100%. n=5. Data are represented asmean+/−SEM). Ad3-PtDd vs. Ad3 knobP=0.0033. b) Competition of Ad3-GFPand Ad35-GFP virus infection. HeLa cells were pretreated with Ad3 fiberknob or PtDd at increasing concentrations and then exposed to Ad3-GFP(left panel) or Ad35-GFP virus (right panel) at an MOI of 100 pfu/cell.GFP expression was measured 18 hours later by flow cytometry. Data arerepresented as mean. Standard deviation was less than 10% for all datapoints. c) Attachment of ³H-labeled Ad3 and Ad35 viruses to human andnon-human cell lines. Y79 and Ramos are human retinoblastoma andlymphoma cells, respectively. CHO cells are Chinese Hamster ovary cells.MMC and TC1 cells are mouse mammary carcinoma and lung carcinoma cells,respectively. TC1-CD46 cells express human CD46. Shown are the averagenumber of viral particels attached per cells. n=5. d and e)Identification of receptor X by affinity capture and MS/MS. Membraneprotein fractions were prepared from HeLa and Ramos cells. Protein blotswere hybridized with Ad5/35++ virions (d) and Ad3 virions or Ad3 PtDd(e). Binding was visualized with polyclonal antibodies against Ad35++knob (d) or Ad3 knob (e) (see also FIGS. 1 f and g). Solubilized HeLacell membrane lysates were also immunoprecipitated with DSG2 mAb 6D8crosslinked with protein A/G plus agarose. Western blot ofimmunoprecipitates was performed with DSG2 monoclonal antibody AH12.2(see antiDSG2-IP). f) MS/MS analysis of the 160 kDa band. Upper panel:Structure of DSG2. EC: extracellular domain, EA: juxtamembraneextracellular anchor domain, TM: transmembrane domain. Lower panel:amino acid sequence of DSG2. Highlighted are the peptide sequencescaptured by MS/MS analysis of the 160 kDa band. The triangles in theDSG2 scheme (top panel) indicate the localization of the identifiedpeptides with regards to the different domains. MS/MS analysis detected14 peptides DSG2 with a high confidence factor (20.8% protein coverageand Sequest cross correlation coefficient scores ranging from 2.6 to 5.5for individual peptides). g-i) Biacore plasmon surface resonance studieswith recombinant human DSG2 immobilized on sensorchips. Ad2, Ad3 and Ad5at 5·10⁹ vp per ml (g), different concentrations of PtDd (h) or PtDd andAd3 fiber knob (i) were injected over the activated surface and responsesignals were collected over the indicated time periods.

FIG. 3 Validation of DSG2 as Ad receptor. “Loss-of-function” studies.

a) Competition of ³H-labeled Ad binding by recombinant DSG2. ³H-Ad3,Ad7, Ad14, Ad14a, Ad11, Ad5 and Ad35 virus were pre-incubated with 6 μgml⁻¹ recombinant human DSG2 protein. Attachment of virus particlesincubated with PBS was taken as 100%. For analysis of Ad11 attachment,cells were also incubated with 50 μg ml⁻¹ of Ad35K on ice for one hourbefore adding of Ad11 virus to block CD46. b) Competition of Adtransduction by recombinant DSG1, DSG2 or DSC2 proteins. c) Competitionof ³H-Ad binding by DSG2-specific antibodies. n=5. PBS vs. 6D8: P=0.013;PBS vs. 8E5: P=0.0014. The specificity of mAbs to different DSG2 domainsis as follows (for a scheme of DSG2, see FIG. 1 f): 20G1 (Pro-peptideregion), 7H9 (Pro/EC1), 13B11 (EC1/EC2), 10D2 (EC1/EC2), 8E5 (EC3), 6D8(EC3/EC4). d) and e) Effect of siRNA-mediated DSG2 downregulation on Adattachment (d) and transduction (e). Shown are mean fluorescenceintensity values. n=5. Note that at 18 hours post-infection, GFP levelswere comparable for Ad35-GFP and Ad5/35-GFP, which allowed us to alsouse the first-generation Ad5/35-GFP vector in further studies. f)Cytolysis of Ad3-GFP infected BT474 cells at day 7 after infection.siRNA transfected cells were infected at adjusted MOIs that allow forcomparable initial transduction rates, i.e. MOI 1.0 pfu per cell and 0.5pfu per cell for DSG2 siRNA and control siRNA treated cells,respectively. Seven days later, viable cells were stained with crystalviolet. Despite the higher virus dose, less killing was seen in cellstransfected with DSG2 siRNA, suggesting the importance of DSG2 inlateral spreading of Ad3. g) Cytolysis of Ad3-GFP infected cells at day7 after infection. siRNA transfected small airway epithelial cells wereinfected at adjusted MOIs. Seven days later, cell viability was measuredby WST-1 assay. Viability of PBS treated cells was taken 100%. n=3.Ad3-GFP/control siRNA vs. Ad3-GFP DSG2 siRNA: P<0.001.

FIG. 4 Validation of DSG2 as Ad receptor. “Gain-of-function” studies.

a) Transduction of human cell lines that express different DSG2 levels.Human erythromyeloblastoid leukemia K562 cells and Burkitt's B-celllymphoma BJAB and Ramos cells were infected with Ad3-GFP and Ad5/35-GFPat increasing MOIs and GFP expression was measured 18 hours later. N=3.Standard deviation was less than 10% for all data points. b) EctopicDSG2 expression. Human histiocytic lymphoma U937 cells were infectedwith a lentivirus vector carrying the DSG2 cDNA under the control of theEF1α, promoter. Stable DSG2 expression was detected in >98% oflentivirus transduced cells by flow cytometry. c) Attachment of ³H-Ad3and ³H-Ad35 to Raji, 0937 and DSG2-expressing 0937 (U937-DSG2) cells.Note that Ad35 attachment is mediated through CD46 and can be blocked bysoluble CD46 (data not shown). d) GFP expression after transduction ofU937 and U937-DSG2 cells with Ad3-GFP and Ad5/35-GFP. n=3

FIG. 5 DSG2 localization in human epithelial cells and interaction withAd3.

a) Immunohistochemistry studies on human colon, foreskin and ovariancancer paraffin sections with DSG2-specific antibodies. Positivestaining appears in brown. b) Confocal microscopy immunofluorescenceanalysis of polarized human colon cancer T84 cells for DSG2 (green) andthe intercellular junction protein Claudin 7 (red). Nuclei are blue. XYand XZ planes are shown. c) Ad3 binding to DSG2. T84 cells wereincubated with Cy3-labeled Ad3 particles (red) for 15 minutes, washed,and subjected to confocal microscopy. The upper XZ image is a highermagnification. Note that at least two (green) DSG2 signals areassociated with one (red) Cy3-Ad3 signal. d) Confocal microscopy ofnormal human small airway epithelial cells (not grown in Transwellchambers). Cells were incubated with Cy5-labelled PtDd for 15 min andthen washed with PBS. The upper XZ panel shows co-localization of DSG2(red) and E-cadherin (green). The lower XZ panel is the same imageshowing purple Cy5-PtDd signals co-localized with green E-cadherinsignals. The XY panel shows purple (PtDd) and green E-cadherin channels.Thin arrows mark membrane localized PtDd, thick arrows label cytoplasmicDSG2. e) Confocal microscopy immunofluorescence analysis of humancervical carcinoma HeLa cells (upper XZ and XY panels) and HeLa cellsincubated for 15 min with Cy3-Ad3 (lower XZ panel). Scale bars for allconfocal microphotographs are 20 μm.

FIG. 6 Epithelial-to-mesenchymal transition signaling induced by Ad3virions and PtDd in epithelial cells.

a-d) Phenotypic changes triggered by PtDd in breast cancer epithelialcells. 1×10⁵ BT474 cells were incubated with 50 ng of PtDd or BsDd forthe indicated time and subjected to staining with antibodies. The scalebar is 20 μm in all ZY confocal images (a, b) and 40 μm in the standardimmunofluorescense studies (c, d). e) Graphic demonstration of arraydata for up- and down-regulated genes (PtDd vs. PBS treated cells). Eachdot represents one gene. f) Western blot analysis of ERK1/2-MAPK andPI3K phosphorylation analyzed 6 hours after incubation of BT474 cellswith PtDd, BsDd, DSG2-specific antibodies (10D2, 13B11, or 6D8), orcontrol antibody (anti-GAPDH) at the indicated concentrations. Forpathway inhibition, cells were treated overnight with Erk1/2 inhibitorUO126 (5 μM) or PI3K inhibitor Wortmannin (2.5 μM) before PtDd wasadded. The efficacy of the drugs for inhibition of the specific pathwaywas validated in a previous study⁸. GAPDH is used to demonstrate equalloading.

FIG. 7 Opening of intercellular junctions in epithelial breast cancercells by interaction of Ad3 virions or PtDd with DSG2.

a) FITC-Dextran diffusion through monolayers of BT474 cells. BT474 cellscultured in transwell chamber with 0.4 μm pore size were treated with0.5 μg ml⁻¹ BsDd, PtDd or 2×10⁸ Ad particles per ml for 2 hours and then4 kDa FITC-dextran was added to the apical compartment. Paracellularflux was assessed in aliquots from the apical and basal chambers. BsDdvs. PtDd: P<0.001. b) Facilitation of ³H-Ad35 uptake by PtDd. Leftpanel: Trapping of CD46 in intercellular junctions of T84 cells.Co-localization of CD46 and the intercellular junction protein Claudin 7results in yellow signals. Right panel: ³H-Ad35 attachment. BT474-cellswere incubated with PtDd or BsDd and ³H-Ad35 for 2 hours on ice, washed,and then incubated at 37° C. for 60 min. Non-internalized Ad particleswere removed by trypsin digestion and cell-associated radioactivity wasmeasured. c) Mice carrying subcutaneous ovc316 tumors were injectedintravenously with 50 μg PtDd or BsDd eight hours before intravenousinjection of 1×10⁹ pfu of Ad5/35-bGal. Sections were stained with X-gal72 hours after injection. The scale bar is 40 μm. d) Confocal microscopyfor Her2/neu and Claudin 7 in the Her2/neu-positive human breast cancercell line BT474. These cells do not form monolayers. Note that in PBStreated cells, most Her2/neu signals (green) colocalize with Claudin 7(red) resulting in yellow signals. Upon PtDd treatment, Claudin 7signals decrease while more Her2/neu staining appears on the cellsurface. e) Confocal microscopy of BT474 cells two hours after treatmentwith PBS or PtDd. f) Ad3 and PtDd enhance killing of Her2/neu positivebreast cancer cells by Herceptin. Viability of PBS-treated cells wastaken 100%. n=5, *P<0.05. g) Ad3 and PtDd enhancement of Herceptintherapy is mediated by DSG2 and involves ERK/MAPK and PI3K pathways.BT474 cells were transfected with control and DSG2 siRNA as described inFIGS. 2 d and 48 hours later treated with Ad3 or PtDd and Herceptin asdescribed in g). For inhibitor studies, BT474 cells were incubated withthe indicated agents overnight. Cells were washed and treated withPtDd/Ad3 and Herceptin as described in above. n=5, PBS vs. Wortmannin,U0126: P<0.05. h) PtDd-mediated enhancement of Herceptin therapy invivo. Shown is the tumor volume of individual mice at different daysafter BT474-M1 cell injection.

FIG. 8. Effect of PtDd on mAb therapy. a) Ad3 and PtDd do not enhancekilling of Her2/neu-negative MDA-MB-231 breast cancer cells byHerceptin. MDA-MB-231 breast cancer cells were incubated with 0.5 mg/mlof BsDd or PtDd, or 2×10⁸ viral particles/ml of uv-inactivated Ad5 orAd3 for 12 hours, followed by an incubation with Herceptin (15 mg/ml)for 30 min. Cell viability was measured 2 hours later by WST-1 assayfrom Roche Biosciences. Viability of PBS-treated cells was taken 100%.b) Ad3 and PtDd enhance killing of EGFR-positive colon cancer cells byErbitux (anti-EGFR). LoVo cells (EGFR-positive) were incubated with 0.5mg/ml of PtDd for 12 hours, followed by incubation with Erbitux (15mg/ml) for 30 min. Cell viability was measured 2 hours later by WST-1assay. Viability of PBS-treated cells was taken 100%. *p<0.05. c) Effectof DSG2 siRNA on adherence junctions of BT474 cells. Shown is claudin 7staining of BT474 cells at day 2 after treatment with PBS or DSG2 siRNA.The scale bar is 40 mm.

FIG. 9. Role of DSG2 in transduction of chimeric Ad5/3 vectors. A)Structure of Ad5/3 vectors. The vectors are based on Ad5 and are deletedfor the E1 and E3 regions. Both vectors contain a GFP expressioncassette inserted into the E3 region. In Ad5/3L-GFP, the Ad5 fiber knobdomain is replaced by that from Ad3. In Ad5/3S-GFP the Ad5 shaft andknob domains were replaced by the corresponding domains from Ad3. TheAd3 shaft domain contains 6 shaft motifs, while the Ad5 shaft domaincontains 22 shaft motifs. B) Blocking of Ad attachment by recombinantDSG2 protein. H³-labeled Ads were incubated with 6 mg/ml of recombinanthuman DSG2 protein on ice for one hour and then added to HeLa cells for1 hour on ice. Ad attachment to cells incubated with PBS instead of DSG2was taken as 100%. n=3, i.e. three separate wells. Ad3-GFP is a vectorderived from Ad3 that contains the same GFP expression cassette as theAd5/3 vectors. C) Blocking of Ad attachment by recombinant DSG2 protein.Ad vectors were incubated with increasing concentrations of DSG2 proteinat room temperature for 60 minutes. Then, HeLa cells were infected at anMOI of 100 pfu/cell for 60 min, after which the viruses were removed andnew medium was added. GFP fluorescence was measured 18 hours later byflow cytometry. n=3. Shown are average values. The standard deviationwas less than 10% for all samples. D) Competition of Ad infection by Ad3PtDd. HeLa cells were incubated with increasing concentrations of PtDdfor 60 minutes and then infected with Ad vectors at an MOI of 100pfu/cell for 60 min, after which the viruses were removed and new mediumadded. GFP fluorescence was measured 18 hours later. N=3. Shown areaverage values. The standard deviation was less than 10% for allsamples. E) DSG2 siRNA blocks infection of Ad5/3 vectors. A total of onemicrogram of siRNA was transfected onto 1×10⁵ HeLa cells. Cells werecollected 48 hours after transfection by Versene, and 1×10⁵ cells werere-plated. The second day, cells were infected with Ad vectors at an MOIof 100 pfu/cell. GFP fluorescence was measured 18 hours later.

FIG. 10. Blocking of Ad3 infection requires cross-linking of Ad3 fiberknobs. A) Structure of a recombinant Ad3 fiber knobs (S6/Kn) containingan N-terminal His-tag, six shaft motifs (S6), and the knob domain (Kn).Additional fiber knob variants contained 5, 4, 3, 2, or 1 shaft motifsand were labeled S5/Kn, S4/Kn, S3/Kn, S2/Kn, and S/Kn, respectively. B)Western blot analysis of recombinant Ad3 fiber knobs. Filters wereincubated with recombinant DSG2, followed by mouse monoclonal anti-DSG2antibodies and anti-mouse IgG HRP conjugates. Visible are trimeric formsof the Ad3 fiber knobs in the range of 50 to 70 kDa. The theoreticalmolecular weights of (timeric) S6/Kn, S5/Kn, S4/Kn, S3/Kn, S2/Kn, andS/Kn are 93.9, 89.7, 84.3, 79.2, 74.4, and 69.3 kDa. S6/Kn and S2/Knformed more multimers of fiber knobs (>75 kDa) than the other knobs andtended to generate inclusion bodies in E. coli. Denaturation of fiberknobs would result in 25-30 kDa monomers (not shown). C) Western blotusing antibodies against the Ad3 fiber knob as a probe. D and E)Competition of Ad3-GFP transduction. HeLa cells were incubated withincreasing concentrations of PtDd and different Ad3 fiber knobs for 60minutes and then infected with Ad3-GFP at an MOI of 100 pfu/cell for 60min, after which the viruses were removed and new medium added. GFPfluorescence was measured 18 hours later. n=3. Shown are average valuesof percent GFP-positive cells (D) and mean GFP fluorescence (E). Thestandard deviation was less than 10% for all samples. S5/Kn is not shownfor clarity. F) Crosslinking of His-tagged fiber knob (S/Kn) by anti-Hisantibodies. Anti-His mAb was incubated with PBS or S/Kn for 15 min andrun on a native polyacrylamide gel. The antibody has a molecular weightof 150 kDa. An additional band with a higher molecular weight appearedin the presence of S/Kn reflecting a complex of both proteins. The knobalone is not shown. G) Effect of cross-linking of Ad3 fiber knobs withanti-His antibodies on inhibition of Ad3-GFP transduction. 5 mg/ml ofAd3 fiber knobs were incubated with 20 mg/ml of mouse anti-His mAbs atroom temperature for 60 minutes, then added onto 1×10⁵ HeLa cell. After60 minutes incubation, 100 pfu/cell of Ad3GFP virus were added and GFPwas analyzed as described in D). The differences between “knob” and“knob+anti-His” were significant (P<0.005) for all Ad3 fiber knobs. Forcomparison, we also included Ad35 fiber knob (non-dimerizing) into thisstudy. H) Effect of cross-linked Ad3 or Ad35 fiber knobs on Ad35-GFPtransduction. Ad35-GFP is a vector derived from Ad35 containing aCMV-GFP expression cassette (33). Ad3 and Ad35 fiber knob are proteinsthat contain a His-tag, one shaft motif, and the corresponding knobs(31). (Ad3 knob is the same as S/Kn). The experiment was performed asdescribed in G). The difference between “knob” and “knob+anti-His” wasnot significant.

FIG. 11. Ad3 fiber knob dimerization via E-/K-coils. A) Schematicstructure of recombinant Ad3 fiber knob proteins, containing anN-terminal His-tag, dimerization domains (E-coli or K-coil (37)), aflexible linker, two fiber shaft motifs (5^(th) and 6^(th)), and the Ad3fiber knob domain. Ad3-S2/Kn is a fiber that lacks the dimerizationdomains. B) Competition of Ad3-GFP transduction. HeLa cells wereincubated with increasing concentrations of PtDd and different Ad3 fiberknobs for 60 minutes and then infected with Ad3-GFP at an MOI of 100pfu/cell for 60 min, after which the viruses were removed and new mediumadded. GFP fluorescence was measured 18 hours later. n=3. Shown areaverage values of percent GFP-positive cells. Ad3-K/S2/Kn+Ad3-E/S2/Kn isa 1:1 mixture of both fiber knobs. PtDd vs Ad3-E/S2/Kn: p=0.074; PtDd vsAd3-K/S2/Kn+Ad3-E/S2/Kn: P=0.03; Ad3-K/S2/Kn vs Ad3-K/S2/Kn+Ad3-E/S2/Kn:p=0.62. C) Cross-linking of Ad3 fiber knobs with anti-His antibodies. 5mg/ml of Ad3 fiber knobs were incubated with 20 mg/ml of mouse anti-HismAbs at room temperature for 60 minutes, then added onto 1×10⁵ HeLacell. After 60 minutes incubation, 100 pfu/cell of Ad3-GFP virus wereadded and GFP was analyzed as described in B). The difference between“knob” and “knob+anti-His” is significant for Ad3-E/S2/Kn (p<0.05), butnot for the other samples.

FIG. 12. Analysis of dimeric fiber knobs containing only one shaftmotif. A) Schematic structure of recombinant Ad3 fiber knob proteinsAd3-K/S/Kn and Ad3-E/S/Kn. The theoretical molecular weight of eachfiber knob trimer is ˜60 kDa. B) Coomassie blue stained gel. Sampleswere run on a 4-15% gradient polyacrylamide gel in Tris/glycine/0.1% SDSbuffer. UB-unboiled samples, B-boiled samples. Note that boiling inLaemmli buffer disrupts the trimeric protein structures resulting in ˜25kDa fiber knob monomers. C) Negative stain electron microscopy ofpurified Ad3-K/S/Kn and Ad3-K/S/Kn mixed with Ad3-E/S/Kn. The upper leftimage shows fiber knob dimers in both preparations. Note that the fiberknob itself is a trimer. The lower images show aggregates that containmore than two fiber knobs. The right panels show schematic drawings ofthe photographs. D) Blocking of ³H-Ad3 attachment by recombinant Ad3fiber knobs or PtDd. Ad attachment to cells incubated with PBS was takenas 100%. N=3. E) Blocking of ³H-Ad5 attachment by recombinant Ad3 fiberknobs or PtDd. Ad attachment to cells incubated with PBS was taken as100%. N=3. F and G) Competition of Ad3-GFP transduction. HeLa cells wereincubated with increasing concentrations of fiber knobs or PtDd for 60minutes and then infected with Ad3-GFP at an MOI of 100 pfu/cell for 60min, after which the viruses were removed and new medium added. GFPfluorescence was measured 18 hours later. n=3. Shown are average valuesof percent GFP-positive cells (F) and mean GFP fluorescence (G). Thestandard deviation was less than 10% for all samples. H and I) The samestudy as in F and G) was performed with Ad3-K/S/Kn and the fiber knobswith two shaft motifs, Ad3-K/S2/Kn.

FIG. 13. SPR analysis of Ad3-K/S/Kn+Ad3-E/S/Kn and Ad3-K/S/Kninteraction with DSG2. A) Biotinylated fiber knobs were immobilized tostreptavidin-linked sensorchips. DSG2 was injected at indicatedconcentrations (3 and 10 mg/ml). Response signals were collected overthe indicated time periods with automatic background subtraction. B)DSG2 was immobilized on sensorchips and background was automaticallysubtracted from the control flowcell. Injection of Ad3 fiber knob(non-dimerizing), Ad3-K/S/Kn and Ad3-E/S/Kn+Ad3-K/S/Kn at 10 mg/ml, andPtDd at 3 mg/ml to normalize all the responses to about 100RU (takinginto account that dodecahedron has 12 fibers and that SPR signal dependson the molecular weight of the analyte.) C) Summary of SPR data shown inB) and calculation of remaining signal 150s after the end of injection.D) Confocal immunofluorescence analysis of DSG2 and Ad3 particles onepithelial colon cancer T84 cells. Shown are cells from the lateralside, i.e. stacked XZ confocal image layers. Cells were exposed to Cy-3labeled Ad3 particles for 15 min, washed, fixed, and stained withanti-DSG2 antibodies (green). Ad3 particles appear in red. The scale baris 20 mm. The right panel shows a schematic drawing of the confocalimage with two DSG2 units clustered by the Ad3 particle. E) XY sectionsof the cell surface and 1 mm deeper. The images suggest that Ad3 bindsto DSG2 molecules that is exposed on the cells surface. Note that mostof the DSG2 is localized, deeper, i.e. distal of tight junctions.

FIG. 14. Analysis of epithelial junctions. Studies were performed onpolarized colon carcinoma T84 cells cultured for 20 days in transwellchambers. A) Confocal immunofluorescence microscopy. Shown arerepresentative stacked XZ images. Upper panel: DSG2 (green) appears atthe apical site of baso-lateral junctions marked by claudin 7 (red).Lower panel: The tight junction marker ZO-1 (red) is localized at theapical side of DSG2 (green). Claudin 7 staining masks the lower part ofDSG2 “streaks” in the lateral membrane, while ZO-1 staining covers theupper part of DSG2 signals. The scale bar is 20 mm. B) Shown are XYsections from the cell surface and 1 mm deeper stained for DSG2 (green)and ZO-1 (red). C) Cells were treated with Ad3-K/S/Kn (5 mg/ml) andanalyzed 12 hours later for DSG2 and ZO-1. D) Transmission electronmicroscopy of junctional areas of T84 cells. Cells were either treatedwith PBS (left panel) or Ad3-K/S/Kn (right panels) for one hour on ice,washed, and then incubated for 1 hour at 37° C. At this time, theelectron-dense dye ruthenium red (1) was added together with thefixative. If tight junctions (above the desmosomes) are closed, the dyeonly stains the apical membrane (black line). If tight junctions areopen, the dye penetrates between the cells and stains the baso-lateralmembrane. The scale bar is 1 mm. Magnification is 40,000×. E) A largermagnification (100,000×) shows the disintegration of desmosomes (markedby an arrow) after Ad3-K/S/Kn treatment. The scale bar is 0.2 mm.

FIG. 15. Functional analyses of epithelial junction opening. A) T84cells were grown in polyester membrane transwell inserts for 21 daysuntil transepithelial resistance was constant, implying that tightintercellular junctions had formed. Shown is ¹⁴C-PEG-4,000 diffusionthrough polarized T84 cells cultured in Ttanswell chambers. Cells wereincubated with PBS or the various DSG2 ligands for 15 min, after which¹⁴C-PEG-4,000 was added to the inner chamber. Paracellular flux wasassessed in aliquots from the apical and basal chambers as describedelsewhere (1). The following monoclonal antibodies against differentextracellular domains (ECD) of DSG2 were used: 13B11-mAb (against theECD 1/2) and 6D8-mAb (against the ECD 3/4). Ad3-K/S/Kn is the dimerizingform. Ad3-E/S/Kn is unable to dimerize. B) Effect of DSG2 ligands ontrastuzumab killing of Her2/neu-positive breast cancer BT474-M1 cells.Cells were incubated at 100% confluence for 2 days. Ligands were addedto the inner chamber for 1 hour followed by PBS or trastuzumab. Cellviability was measured two hours later (see Material and Methods). Inaddition to 13B11 and 6D8 mAbs, the following anti-DSG2 antibodies wereused: 7H9 (against the pro-peptide domain), 10D2 (against the ECD 2),8E5 against the ECD 3/4). Viability of dilution buffer-treated cells wastaken 100%. n=5, i.e. five separate wells. *: p<0.05 compared todilution buffer. C) Confocal microscopy for CAR in T84 cells treatedwith dilution buffer or Ad3-K/S/Kn (stacked XZ layers). T84 cells weregrown in polyester membrane transwell inserts for 21 days. Ad3-K/S/Kn(40 mg/ml) or dilution buffers were added for 60 min, after which cellswere washed and subjected to immunofluorescence analysis. CAR appears asgreen staining. Nuclei are blue. The scale bar is 20 mm. D) Confocalmicroscopy for CAR in polarized T84 cells. XY images were taken of thecell surface and at a layer 1 mm beneath the cell surface (rightpanels). Experimental conditions were as in C). The scale bar is 20 mm.E) Ad transduction of T84 cells. T84 cells were grown in polyestermembrane transwell inserts for 21 days. Ad3-GFP and Ad5-GFP was added tothe inner chamber at an MOI of 250 pfu/cell together with dilutionbuffer (upper panels) or Ad3-K/S/Kn (40 mg/ml). Three hours later, viruswas removed and cells were washed. GFP expression was analyzed after 20hours of incubation. Shown are representative images. Forquantification, GFP-positive cells from 10 independent images of threeindependent experiments were counted. The scale bar is 20 mm. F) Flowcytometry of Ad5-GFP infected cells. T84 cells were infected withAd5-GFP as described in E) either in the presence of dilution buffer or40 mg/ml DSG2 ligands. n=3. *: P<0.05 compared to dilution buffer.

FIG. 16. Transient opening of epithelial junctions by JO-1. A) Structureof Ad3 viral particles. Left panel: complete, infectious Ad3 particle.The capsid proteins fiber and penton base are shown in green and blue,respectively. The trimeric fiber knob is shown in red. Middle panel: Ad3pentondodecahedra (PtDd) formed by spontaneous assembly of 12recombinant pentons (fiber+penton base). Right panel: dimeric Ad3 fiber(JO-1). B) Schematic structure of JO-1 containing an N-terminal His-tag,a dimerization domain [K-coil (Zeng et al., 2008)], a flexible linker,one fiber shaft motif, and the homotrimeric Ad3 fiber knob domain. JO-1is produced in E-coli (at a yield of ˜10 mg/l) and purified withNi-columns. C) Left panel: simplified structure of epithelial junctionswith tight junctions, desmosomes, and adherens junctions. DSG2 is adesmosomal protein. Claudin 7 is an adherens junction protein. Rightpanels: Confocal immunofluorescence microscopy of T84 cells. Shown arerepresentative stacked XZ images. Cells were treated with JO-1 (5 μg/ml)for 1 h on ice. After removal of JO-1, cells were incubated at 37° C.and analyzed 0, 30, and 60 min later. Upper panel: DSG2 (green) appearsat the apical site of baso-lateral junctions marked by claudin 7 (red).Middle panel: within 30 min after adding JO-1, claudin 7 stainingincreases and DSG2 staining becomes visible along the upper part of thelateral membrane (yellow signals). Lower panel: By 60 minutes, lateraljunctions resemble those of time point “0 min”. The scale bar is 40 μm.D) Transmission electron microscopy of junctional areas of polarizedcolon cancer T84 cells. Cells were either treated with PBS (left panel)or JO-1 (right panel) for 1 h on ice, washed, and then incubated for 1 hat 37° C. At this time, the electron-dense dye ruthenium red (Amieva etal., 2003) was added together with the fixative. If tight junctions(above the desmosomes, marked by arrows) are closed, the dye only stainsthe apical membrane (black line). If tight junctions are open, the dyepenetrates between the cells and stains the baso-lateral membrane. Thescale bar is 1 μm. Magnification is 40,000×. E) ¹⁴C-PEG-4,000 diffusionthrough monolayers of T84 cells at different time points after addingJO-1 or anti-DSG2 antibody (6D8, directed against ECD3/4). Cells wereincubated on ice for 1 h with DSG2 ligands and washed. Fresh mediumcontaining ¹⁴C-PEG-4,000 was then added to the inner chamber.Paracellular flux was assessed in aliquots from the apical and basalchambers as described elsewhere (Amieva et al., 2003). The experimentwas repeated 3 times.

FIG. 17. Analysis of mechanism of JO-1 action in tumors in vivo. A totalof 4×10⁶ human breast cancer HCC1954 cells were injected into themammary fat pad of CB17-SCID/beige mice. Thirty days later, when tumorsreached a volume of ˜200 mm³, JO-1 (2 mg/kg in 200 μl PBS) was injectedintravenously. Tumors were harvested either 1 or 12 h after JO-1injection. Control mice received 200 μl PBS and tumors were collected 1h later. A) Kinetics of JO-1 accumulation in tumors. Left panels:immunofluorescence analysis of tumor sections using anti-His tagantibodies (for visualization of JO-1). Representative sections areshown. The scale bar is 20 μm. Right panel: Western blot analysis oftumor tissue using Ad3-fiber knob specific antibodies (Wang et al.,2011b). The specific band representing JO-1 is marked by an arrow.Representative images are shown. The experiment was repeated 3 times. B)Analysis of DSG2 in tumors. Left panel: immunofluorescence analysis oftumor sections using DSG2 antibodies (mAb 6D8 against extracellulardomain 3/4 of DSG2). The inserts show a higher magnification. Rightpanel: The same anti-DSG2 antibody was used for Western blot analysis oftumor tissue. C) Intracelluar signaling in vivo. Left panel: Westernblot analysis of tumor tissue for E-cadherin and phosphorylatedE-cadherin, Erk 1/2, phosphorylated Erk1/2, claudin 7, and vimentin.Antibodies against gamma-tubulin were used to assess sample loading(“loading control”). Right panels: immunofluorescence analysis usingantibodies against E-cadherin and phosphorylated Erk1/2. Representativeimages are shown. The experiment was repeated 3 times.

FIG. 18. JO-1 improves penetration of trastuzumab in HCC1954 breastcancer tumors in situ. Tumor bearing mice were intravenously injectedwith PBS or JO-1 (2 mg/kg) followed by trastuzumab 1 h later. Tumorswere harvested 1 h or 12 h after trastuzumab injection. A) Sections werestained for human IgG (i.e. trastuzumab). Positive staining appearsgreen. Representative sections are shown. The scale bar is 20 μm. B)Western blot analysis for human IgG (trastuzumab) in tumors. Heavy (HC)and light (LC) Ig chains are indicated by arrows. Ponceau S staining fortotal protein blotted to the membrane serves a loading control.Representative images are shown.

FIG. 19. JO-1 increases mAb killing of cells in which the targetreceptors are trapped in epithelial junctions. A) Confocal microscopy ofHer2/neu (green) and DSG2 (red) staining on polarized BT474 cellcultures (XY and XZ images). Cells treated with PBS are shown in theleft panel. Middle and right panels: Cells were treated with JO-1 (20μg/ml) for 1 h on ice. After removal of JO-1, cells were incubated at37° C. and analyzed 1 h and 16 h later. XY images show the cell surface(left) and a section 2 μm below the cell surface. The scale bar is 40μm. B) Confocal microscopy of EGFR (red) and the tight junction proteinE-cadherin (green) on polarized A549 lung cancer cells. C) JO-1 enhanceskilling of Her2/neu-positive breast cancer cells by trastuzumab. BT474cells were incubated with JO-1 (5 μg/ml) or PBS. Trastuzumab (15 μg/ml)was added 1 h later. Cell viability was measured after 3 h by WST-1assays as described earlier (Wang et al., 2010). Viability ofPBS-treated cells was taken as 100%. n=5, P<0.05 for trastuzumab vs.JO-1+trastuzumab. Representative images are shown. The experiment wasrepeated 3 times. D) JO-1 enhances cetuximab killing of EGFR-positiveA549 cells. n=5, P<0.05 for cetuximab vs. JO-1+cetuximab.

FIG. 20. JO-1 improves trastuzumab therapy in Her2/neu positive breastcancer models. A) A total of 4×10⁶ BT474-M1 cells, (a tumorigenicsubclone of BT474) were injected into the mammary fat pad ofCB17-SCID/beige mice. 29 days later (when tumors reached a volume of˜100 mm³), mice received an intravenous injection of 50 μg JO-1 (2mg/kg) or PBS, followed by an intraperitoneal injection of trastuzumab(10 mg/kg)* or PBS 10 h later. A second treatment cycle was started atday 36 (marked by arrows). Shown is the increase in tumor volume(compared to pretreatment levels at day 29) in individual animals. n=5.Tumor volumes at day 40 were significantly lower in JO-1+trastuzumabtreated mice than in animals that received trastuzumab alone (P<0.01).*This dose of trastuzumab and its route of application is routinely usedin mice (Beyer et al., 2011). B) A similar therapy study was performedwith HCC1954 Her2/neu-positive breast cancer derived tumors. Thesetumors are more resistant to trastuzumab treatment. A total of 4×10⁶HCC1954 cells, were injected into the mammary fat pad of CB17-SCID/beigemice. Eighteen days later (when tumors reached a volume of ˜100 mm³),mice received an intravenous injection of 50 μg JO-1 (2 mg/kg) or PBS,followed by an intraperitoneal injection of trastuzumab or PBS 10 hlater. n=5. trastuzumab vs. JO-1+trastuzumab P<0.001. C) Effect ofvarious time intervals between JO-1 and trastuzumab injection ontherapeutic outcome. Mice bearing HCC1954 breast cancer tumors wereinjected with a mixture of JO-1 and trastuzumab, JO-1 followed bytrastuzumab 1 h later and, JO-1 followed by trastuzumab 10 h later.Injections were repeated weekly. n=5 trastuzumab vs. JO-1+trastuzumab(10 h) P<0.001; trastuzumab vs. JO-1+trastuzumab (1 h) P<0.0058;trastuzumab vs. JO-1+trastuzumab (mixed) P<0.0074. JO-1+trastuzumab (10h) vs. JO-1+trastuzumab (1 h) P<0.17, not significant (ns);JO-1+trastuzumab (10 h) vs. JO-1+trastuzumab (mixed) P<0.47, ns.

FIG. 21. JO-1 improves trastuzumab therapy in a Her2/neu-positivegastric cancer model. left panel: Immunofluorescence microscopy ofHer2/neu-positive human gastric cancer cells (NCI-N87). The scale bar is20 μm. right panel: JO-1/trastuzumab treatment of mice bearingsubcutaneous human gastric cancer (NCI-N87) xenograft tumors. n=5,trastuzumab vs. JO-1+trastuzumab (days 29 and 33): P<0.05

FIG. 22. JO-1 improves cetuximab therapy in lung cancer xenograftmodels. A) Immunodeficient CB17-SCID/beige mice were subcutaneouslyinjected with 1×10⁶ A549 cells. JO-1 was injected at day 11intravenously (2 mg/kg) or intraperitoneally (4 mg/kg) followed by anintraperitoneal injection of cetuximab or PBS 10 h later. One groupreceived 1 mg/kg of JO-1 intravenously and 1 mg/kg intratumorally. n=5,cetuximab vs. JO-1 i.v.+i.t. plus cetuximab P<0.001; cetuximab vs. JO-1i.v. plus cetuximab P<0.001; cetuximab vs. JO-1 i.p.+cetuximab P<0.001.The differences between the different JO-1 injection routes were notsignificant. B) Immunodeficient CB17-SCID/beige mice were subcutaneouslyinjected with 1×10⁶ A549 cells. Eleven days later (when tumors reached avolume of ˜100 mm³), mice received an intravenous injection of 2 mg/kgPtDd followed by an intraperitoneal injection of cetuximab (10 mg/kg) orPBS 10 h. A second treatment cycle was started at day 15 (marked byarrows). n=5. Cetuximab vs. PtDd plus cetuximab P<0.001. C) Metastaticlung cancer model. CB17-SCID/beige mice were intravenously injected with1×10⁶ A549 cells. 10 days later, mice received an intravenous injectionof 2 mg/kg JO-1 or PBS, followed by an intraperitoneal injection ofcetuximab (10 mg/kg) or PBS 10 h later. The treatment was repeated every3 days until day 38. n=10. Left panels: Lungs from individual micestained with India ink. Healthy tissue appears black. Tumor tissuestains white. Right panel: representative sections of lungs stained withH&E.

FIG. 23. Combination therapy of JO-1 and relaxin in the HCC1854 breastcancer model. A) Schematic illustration of the experiment. Lethallyirradiated mice received either mock transduced or LV-EF1a/RIxtransduced Lin⁻ hematopoietic stem cell. Six weeks later, afterengraftment of HSCs, mice were injected into the mammary fat pad with4×10⁶ HCC1954 cells. Relaxin expression was activated by Doxycyclin 7days later. Mice were then given weekly treatment of PBS,PBS/trastuzumab or JO-1/trastuzumab and tumor volumes were measured. B)Tumor volumes of individual mice. n=5. trastuzumab vs. JO-1+trastuzumabP<0.001: RIx JO-1+trastuzumab vs. RIxtrastuzumab P<0.001; RIx PBS vs.PBS P<0.001.

FIG. 24 Alignment of the fiber polypeptides of Ad3, Ad7, Ad11, Ad14, andAd14a and their domain structure is noted.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N. Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E J.Murray, The Humana Press Inc., Clifton, N. J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine(Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

As used herein, the abbreviation “Ad” refers to an adenovirus and istypically followed by a number indicating the serotype of theadenovirus. For example, “Ad3” refers to adenovirus serotype 3.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the invention provides methods for enhancingtherapeutic treatment, diagnosis, or imaging of a disorder associatedwith epithelial tissue, comprising administering to a subject in needthereof (a) an amount of one or more therapeutics sufficient to treatthe disorder, diagnostic sufficient to diagnose the disorder, and/orimaging agent sufficient to image the epithelial tissue; and (b) anamount of AdB-2/3 fiber multimer, or functional equivalent thereof,sufficient to enhance efficacy of the one or more therapeutics,diagnostics, and/or imaging agents.

The methods of this aspect of the invention can be used to enhancingtherapeutic treatment, diagnosis, or imaging of a disorder associatedwith epithelial tissue by improving access for the therapeutic,diagnostic, and/or imaging agent to their target and dissemination inepithelial tissue. While not being bound by any mechanism, the inventorsbelieve this occurs through complementary mechanisms: movement of thetarget receptor from the basolateral to the apical cell surface thusallowing better access to the epithelial tissue target by therapeutics,diagnostics, and/or imaging agents that target the receptor, such asmonoclonal antibodies), and better penetration of the therapeuticthrough disruption of intercellular junctions. As disclosed in detailherein, the inventors have discovered that desmogelin-2 (DSG2) is theprimary high affinity receptor for AdB-2/3. DSG2 is a calcium-bindingtransmembrane glycoprotein belonging to the cadherin protein family. Inepithelial cells, DSG2 is a component of the cell-cell adhesionstructure. Its cytoplasmic tail interacts with a series of proteins thatare in direct contact with regulators of cell adhesion and intercellularjunctions/cell morphology. It has been shown that DSG2 is overexpressedin a series of epithelial malignancies including gastric cancer,squamous cell carcinomas, melanoma, metastatic prostate cancer, andbladder cancer.

While not being bound by a specific mechanism of action, the inventorsbelieve that the AdB-2/3 fiber multimer binding to DSG2 serves totrigger transient DSG2-mediated opening of intercellular junctions,which serves to improve access of therapeutics, diagnostics, imagingagents, or any other compound of interest that binds to a target inepithelial cells that would otherwise be trapped to at least some extentin intercellular junctions. Detailed examples of such activity areprovided herein.

The methods of the invention have broad application for delivery of anytherapeutic, diagnostic, imaging agent, or other compound to epithelialtissue comprising intercellular junctions where access to a target ofinterest can be limited, as DSG2 is widely expressed in epithelialcells. As used herein, a “disorder associated with epithelial tissue” isany disorder wherein therapeutic, diagnostic, or imaging agentadministered to/across epithelial cells/epithelial tissue provides aclinical benefit to a patient, whether in improving therapeutic,diagnostic, and/or imaging efficacy. Such disorders include, but are notlimited to, solid tumors (i.e.: any tumor with epithelial celljunctions), gastrointestinal disorders (including but not limited toirritable bowel syndrome, inflammatory bowel disorder, Crohn's disease,ulcerative colitis, constipation, gastroesophageal reflux disease,Barrett's esophagus, etc.), skin diseases (including but not limited topsoriasis and dermatitis), lung disorders (including but not limited tochronic obstructive pulmonary disease, asthma, bronchitis, pulmonaryemphysema, cystic fibrosis, interstitial lung disease, pneumonia,pancreatic duct disorders, brain disorders (ie: any brain disorder thatcould benefit from improved transport of drugs through the blood-brainbarrier), primary pulmonary hypertension, pulmonary embolism, pulmonarysarcoidosis, tuberculosis, etc.), renal disorders, (including but notlimited to glomerulonephritis), liver diseases (including but notlimited to hepatitis), endocrine disorders (including but not limited todiabetes and thyroid disorders), pancreatic duct disorders (includingbut not limited to pancreatitis), and bile duct disorders (including butnot limited to bile duct obstruction, cholecystitis,choledocholithiasis, gallstones, etc.) and infections of epithelialtissues (including but not limited to cellulitis, pneumonia, hepatitis,and pyelonephritis). In one preferred embodiment, the disorderassociated with epithelial tissue comprises a solid tumor, including butnot limited to breast tumors, lung tumors, colon tumors, rectal tumors,skin tumors, endocrine tumors, stomach tumors, prostate tumors, ovariantumors, uterine tumors, cervical tumors, kidney tumors, melanomas,pancreatic tumors, liver tumors, brain tumors, head and neck tumors,nasopharyngeal tumors, gastric tumors, squamous cell carcinomas,adenocarcinomas, bladder tumors, and esophageal tumors. As will beunderstood by those of skill in the art, such tumors include primarytumors, tumors that are locally invasive, as well as tumors that havemetastasized.=

As used herein, “enhancing efficacy” means any increase in therapeutic,diagnostic, and/or imaging efficacy over what would be seen using thetherapeutic, diagnostic, and/or imaging agen alone. For example,measurements of therapeutic efficacy will vary depending on the disorderbeing treated, but are readily identified by an attending physician. Forexample, such increases in efficacy include, but are not limited toincreasing one or more of the following relative to treatment with thetherapeutic alone: (a) reducing the severity of the disorder; (b)limiting or preventing development of symptoms characteristic of thedisorder(s) being treated; (c) inhibiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting orpreventing recurrence of the disorder(s) in patients that havepreviously had the disorder(s); and (e) limiting or preventingrecurrence of symptoms in patients that were previously symptomatic forthe disorder(s). In one non-limiting example, treating a solid tumorprovides an ability to induce egress of tumor receptors from thebasolateral side of epithelial cells to enable improved access andkilling of the tumor.

For cancer, there are standards for defining tumor response and standardmethods of measuring response. These include tumor response, which isdetermined by monitoring the change in tumor size or a serum marker ofdisease. A partial response is more than a 50% reduction in the tumor,while a complete response is defined as complete disappearance of thetumor. Methods used to measure tumors are well known to physicians andinclude physical examination, radiological testing such as CT scans,MRI, PET scans, X-rays as well as serum markers such as prostatespecific antigen, which is used to monitor prostate cancer. Othermeasures of therapeutic efficacy of cancer treatment includemeasurements of time to progression, progression-free survival andoverall survival.

Improved diagnostic efficacy includes any improvement in efficacycompared to administration of the diagnostic alone, including but notlimited to, increasing specificity and/or sensitivity of the diagnostictest. Improved imaging efficacy includes any improvement in efficacycompared to administration of the imaging agent alone, including but notlimited to specificity, sensitivity, reproducibility, contrastenhancement, detection of smaller sites of disease, more accuratedelineation of disease, such as size and shape of diseases, such astumors, abscesses, etc.

In various embodiments, the increase in efficacy is a 5%, 10%, 15%, 20%,25%, 50%, 75%, 100%, or greater benefit compared to efficacy with thetherapeutic, diagnostic, and/or imaging agent alone across a patientpopulation.

Any suitable subject can be treated using the methods of the invention,preferably human subjects.

As used herein, “AdB-⅔” is any adenovirus serotype that uses DSG2 as anepithelial cell receptor for viral binding. To date, Ad3, Ad7, Ad11,Ad14, and Ad14a serotypes have been identified. As other Ad serotypesare identified, those of skill in the art can readily identify thosethat belong to the AbD-2/3 family based on DSG2 binding assays asdisclosed herein. For example, surface plasmon resonance (SPR) studiesusing sensors containing immobilized recombinant DSG2 can be used todetermine if new Ad serotypes bind to DSG2, combined with DSG2competition studies. Further exemplary studies, such as loss and gain offunction analyses, are described in detail in Example 1.

The adenovirus virion is an icosahedron characterized by a fiber locatedat the base of each of the 12 vertices of the capsid. The fiber on thevirion is a homotrimeric structure consisting of 3 individual fiberpolypeptides. Each adenovirus fiber polypeptide is an asymmetricalstructure consisting of an N-terminal tail, which interacts with thepenton base protein of the capsid and contains the signals necessary fortransport of the protein to the cell nucleus; a shaft, which contains anumber of 15-residue repeating units; and a C-terminal knob domain thatcontains the determinants for receptor binding (J. S. Hong and J. A.Engler, Journal of Virology 70:7071-7078 (1996)). All adenovirusesattach to their receptors through the knob structure on the end of thefiber.

Thus, as used herein, the term AdB-2/3 “fiber polypeptide” refers thefull length fiber polypeptide expressed by AdB-2/3 (for example, SEQ IDNOS 15-19, with Ad3 fiber polypeptide as SEQ ID NO:15, Ad7 fiberpolypeptide as SEQ ID NO:16, Ad11 polypeptide as SEQ ID NO:17, Ad14fiber polypeptide as SEQ ID NO:18, and Ad14(a) fiber polypeptide as SEQID NO:19) that comprises an N-terminal tail domain, a shaft domain, anda C-terminal knob domain. The fiber polypeptides spontaneously assembleinto homotrimers, referred to as “fibers,” which are located on theoutside of the adenovirus virion at the base of each of the twelvevertices of the capsid.

As used herein, an “AdB-2/3 fiber multimer” is any construct comprisinga multimer (dimer, trimer, etc.) of an AdB-2/3 fiber, or functionalequivalent thereof. As will be understood by those of skill in the art,the AdB-2/3 fiber comprises a homotrimeric knob. The AdB-2/3 fibermultimer is a multimer, such as a dimer, of the homotrimeric AdB-2/3fiber. As disclosed in detail in the examples that follow, AdB-2/3 fibermultimers are required for binding to DSG2 that triggers transientDSG2-mediated opening of intercellular junctions, which serves toimprove access of therapeutics to therapeutic targets that wouldotherwise be trapped in intercellular junctions

In one embodiment, the AdB-2/3 fiber multimer is selected from the groupconsisting of an Ad3 fiber multimer, an Ad7 fiber multimer, an Ad11fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer,combinations thereof, and functional equivalents thereof. In a preferredembodiment the AdB-2/3 fiber multimer is an Ad3 fiber multimer.

Exemplary constructs comprising one or more AdB-2/3 fiber multimers (orchimeras/functional equivalents thereof) include, but are not limitedto, AdB-2/3 virions (such as “killed” virions, for example UV-treatedvirions), AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd)(subviral dodecahedral particles produced by AdB-2/3 during theirreplication), recombinant AdB-2/3 fiber multimers (including but notlimited to those disclosed in any embodiment or combination ofembodiments below), and functional equivalents thereof. In a preferredembodiment, the one or more AdB-2/3 fiber multimers comprise or consistof an AdB-2/3 PtDd, such as an Ad3 PtDd. In another preferredembodiment, the one or more AdB-2/3 fiber multimers comprise or consistof any embodiment or combination of embodiments of the compositions ofthe invention described below. In a further preferred embodiment, theAdB-2/3 fiber multimer comprises or consists of the polypeptide referredto herein as JO-1 (junction-opener-1), (SEQ ID NO:20), or functionalequivalents thereof. As shown in the examples that follow, JO-1(described in the examples as a self-dimerizing Ad3 fiber derivative) isa multimer of a single chain, recombinant AdB-2/3 fiber polypeptidedomain, wherein the polypeptide comprises a knob domain capable ofhomotrimerization. Thus, JO-1 is an AdB-2/3 fiber multimer according tothe present invention.

Methods for preparing large quantities of AdB-2/3 virions and capsid arewell known in the art, as are methods for large scale production ofPtDd. Similarly, methods for recombinant production of AdB-2/3 fibermultimers (such as JO-1) by incorporating dimerization domains in therecombinant polypeptides are well within the level of skill in the artbased on the teachings herein.

The methods of the invention can thus be carried out using any AdB-2/3fiber multimer capable of binding to DSG2 and triggering transientDSG2-mediated opening of intercellular junctions. Thus, non-naturallyoccurring modifications (deletions, additions, substitutions, chimerasof different Ad serotype fiber proteins and domains thereof, etc.) tothe AdB-2/3 fiber multimers disclosed herein are “equivalents thereof”of the AdB-2/3 fiber multimers and are within the scope of the presentinvention, so long as they function to bind DSG2 and are capable ofmultimerizing and triggering DSG2-mediated opening of intercellularjunctions. Based on the teachings herein for testing binding to DSG2 andfor assessing DSG2-mediated opening of intercellular junctions, it iswell within the level of skill in the art to identify such functionalequivalents of the AdB-2/3 fiber multimers. In one non-limiting example,assays to assess the flux of a labeled compound (such as FITC-dextran)through confluent polarized epithelial cells, in the presence ofcandidate AdB-2/3 fiber multimers, are demonstrated in Examples 1 and 2below. In another non-limiting example, assays in the presence ofcandidate AdB-2/3 fiber multimers to assess access to proteins notnormally accessible due to epithelial cell junctions (such as CD46,Claudin 7, and ZO-1) are demonstrated in Examples 1 and 2 below. Furthersuch assays are also disclosed herein.

AdB-2/3 fiber multimerization can be determined according to methodswell known to the practitioners in the art. For example, multimerizationof the recombinant AdB-2/3 fiber constructs can be assessed by criteriaincluding sedimentation in sucrose gradients, resistance to trypsinproteolysis, and electrophoretic mobility in polyacrylamide gels (Hongand Engler, Journal of Virology 70:7071-7078 (1996)). Regardingelectrophoretic mobility, the fiber multimer is a very stable complexand will run at a molecular weight consistent with that of a multimerwhen the sample is not boiled prior to SDS-PAGE. Upon boiling, however,the multimeric structure is disrupted and the protein subsequently runsat a size consistent with the protein monomer. Any therapeutic,diagnostic, imaging agent, or other compound that can target epithelialtissue and whose delivery to epithelial tissue can be improved bytransient opening of intercellular junctions can be used in the methodsof the invention. In one embodiment, the therapeutic is selected fromthe group consisting of antibodies, immunoconjugates, nanoparticles,nucleic acid therapeutics, and combinations thereof, chemotherapeutics,vaccines, radioactive particle/radiation therapy (“radiation”), cellularimmunotherapy including adoptive T-cell therapy and dendritic celltherapy (example: intratumoral penetration of administered T-cells),inhaled therapeutics, gene therapy constructs (including but not limitedto AdB-2/3 virus as a gene therapy vector, and co-administration with anAd5-based gene therapy vector), other nucleic acid therapeutics, andcombinations thereof.

In various embodiments, the therapeutic is selected from the groupconsisting of alkylating agents, angiogenesis inhibitors, antibodies,antimetabolites, antimitotics, antiproliferatives, aurora kinaseinhibitors, apoptosis promoters (for example, Bcl-xL, Bcl-w and Bfl-1)inhibitors, activators of death receptor pathway, Bcr-Abl kinaseinhibitors, BiTE (Bi-Specific T cell Engager) antibodies, biologicresponse modifiers, cyclin-dependent kinase inhibitors, cell cycleinhibitors, cyclooxygenase-2 inhibitors, growth factor inhibitors, heatshock protein (HSP)-90 inhibitors, demethylating agents, histonedeacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,inhibitors of apoptosis proteins (IAPs) intercalating antibiotics,kinase inhibitors, mammalian target of rapamycin inhibitors, microRNA'smitogen-activated extracellular signal-regulated kinase inhibitors,multivalent binding proteins, non-steroidal anti-inflammatory drugs(NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP)inhibitors, platinum chemotherapeutics, polo-like kinase (Plk)inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs,receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids,small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitorsand the like.

Exemplary therapeutics falling within these various classes include, butare not limited to: docetaxel, doxorubicin, irinotecan, paclitaxel(Taxol®), paclitaxel albumin bound particles (Abraxane®), doxorubicinHCL liposome (Doxil®), BiTE antibodies such as adecatumumab (MicrometMT201), blinatumomab (Micromet MT103) and the like, siRNA-basedtherapeutics, alkylating agents including altretamine, AMD-473, AP-5280,apaziquone, bendamustine, brostallicin, busulfan, carboquone, carmustine(BCNU), chlorambucil, CLORETAZINE.® (laromustine, VNP 40101M),cyclophosphamide, dacarbazine, decitabine, 5′-azacytidine, estramustine,fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU),mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogenmustard N-oxide, ranimustine, temozolomide, thiotepa, TREANDA®(bendamustine), treosulfan, rofosfamide and the like; angiogenesisinhibitors including endothelial-specific receptor tyrosine kinase(Tie-2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors,insulin growth factor-2 receptor (IGFR-2) inhibitors, matrixmetalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9(MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR)inhibitors, thrombospondin analogs, vascular endothelial growth factorreceptor tyrosine kinase (VEGFR) inhibitors and the like;antimetabolites including ALIMTA® (pemetrexed disodium, LY231514, MTA),5-azacitidine, XELODA® (capecitabine), carmofur, LEUSTAT® (cladribine),clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside,decitabine, deferoxamine, doxifluridine, eflornithine, EICAR(5-ethynyl-1-.beta.-D-ribofuranosylimidazole-4-carboxamide),enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or incombination with leucovorin, GEMZAR® (gemcitabine), hydroxyurea,ALKERAN® (melphalan), mercaptopurine, 6-mercaptopurine riboside,methotrexate, methotrexate analogs (such as trimetrexate andpralatraxate), mycophenolic acid, nelarabine, nolatrexed, ocfosfate,pelitrexol, pentostatin, raltitrexed, Ribavirin, triapine, trimetrexate,S-1, tiazofurin, tegafur, TS-1, vidarabine, and the like; Bcl-2 proteininhibitors including AT-101 ((−)gossypol), GENASENSE® (G3139 oroblimersen (Bcl-2-targeting antisense oligonucleotide)), IPI-194,IPI-565,N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobe-nzenesulfonamide)(ABT-737),N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide (ABT-263),GX-070 (obatoclax) and the like; Bcr-Abl kinase inhibitors includeDASATINIB® (BMS-354825), GLEEVEC® (imatinib) and the like; CDKinhibitors including AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584,flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib(CYC-202, R-roscovitine), ZK-304709 and the like; EGFR inhibitorsincluding ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine, EMD-7200,ERBITUX® (cetuximab), HR3, IgA antibodies, IRESSA® (gefitinib), TARCEVA®(erlotinib or OSI-774), TP-38, EGFR fusion protein, TYKERB® (lapatinib)and the like; ErbB2 receptor inhibitors include CP-724-714, CI-1033(canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib), OMNITARG®(2C4, petuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974, EKB-569,PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neubispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecificantibodies, mAb AR-209, mAb 2B-1 and the like; histone deacetylaseinhibitors include romidepsin, LAQ-824, MS-275, trapoxin,suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like;HSP-90 inhibitors including 17-AAG-nab, 17-AAG, CNF-101, CNF-1010,CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, MYCOGRAB® (humanrecombinant antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol,SNX-2112, STA-9090 VER49009 and the like; activators of death receptorpathways including TRAIL, antibodies or other agents that target TRAILor death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab,ETR2-ST01, GDC0145, (lexatumumab), HGS-1029, LBY-135, PRO-1762 andtrastuzumab; platinum chemotherapeutics include cisplatin, ELOXATIN®(oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN®(carboplatin), satraplatin, picoplatin and the like; VEGFR inhibitorsincluding AVASTIN® (bevacizumab), ABT-869, AEE-788, axitinib (AG-13736),AZD-2171, CP-547,632, IM-862, MACUGEN (pegaptamib), NEXAVAR® (sorafenib,BAY43-9006), pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584),SUTENT® (sunitinib, SU-11248), VEGF trap, ZACTIMAThi (vandetanib,ZD-6474) and the like; dendritic cell therapy (sipuleucel-T, Provenge®);topoisomerase inhibitors including aclarubicin, 9-aminocamptothecin,amonafide, amsacrine, becatecarin, belotecan, BN-80915, CAMPTOSAR®(irinotecan hydrochloride), camptothecin, dexrazoxine, diflomotecan,edotecarin, ELLENCE® or PHARMORUBICIN® (epirubicin), etoposide,exatecan, abraxane, irenotecan, 10-hydroxycamptothecin, gimatecan,lurtotecan, mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan,sobuzoxane, SN-38, tafluposide, topotecan and the like; antibodiesincluding AVASTIN® (bevacizumab), CD40-specific antibodies, chTNT-1/B,denosumab, ERBITUX® (cetuximab), HUMAX-CD4® (zanolimumab), IGF IR-specific antibodies, lintuzumab, PANOREX® (edrecolomab), RENCAREX® (WXG250), RITUXAN® (rituximab), ticilimumab, trastuzimab and the like;hormonal therapies including ARIMIDEX® (anastrozole), AROMASIN®(exemestane), arzoxifene, CASODEX® (bicalutamide), CETROTIDE®(cetrorelix), degarelix, deslorelin, DESOPAN® (trilostane),dexamethasone, DROGENIL® (flutamide), EVISTA® (raloxifene), AFEMA®(fadrozole), FARESTON® (toremifene), FASLODEX® (fulvestrant), FEMARA®(letrozole), formestane, glucocorticoids, HECTOROL® (doxercalciferol),RENAGEL® (sevelamer carbonate), lasofoxifene, leuprolide acetate,MEGACE® (megesterol), MIFEPREX® (mifepristone), NILANDRON® (nilutamide),NOLVADEX® (tamoxifen citrate), PLENAXIS® (abarelix), prednisone,PROPECIA® (finasteride), rilostane, SUPREFACT® (buserelin), TRELSTAR®(luteinizing hormone releasing hormone (LHRH)), VANTAS® (Histrelinimplant), VETORYL® (trilostane or modrastane), ZOLADEX® (fosrelin,goserelin) and the like; immunologicals including interferon alpha,interferon alpha-2a, interferon alpha-2b, interferon beta, interferongamma-1a, ACTIMMUNE® (interferon gamma-1b) or interferon gamma-n1,combinations thereof and the like. Other agents include ALFAFERONE®(IFN-alpha), BAM-002 (oxidized glutathione), BEROMUN® (tasonermin),BEXXAR® (tositumomab), CAMPATH® (alemtuzumab), CTLA4 (cytotoxiclymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE®(lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010(anti-CTLA-4), melanoma vaccine, mitumomab, molgramostim, MYLOTARG™(gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL, OVAREX®(oregovomab), pemtumomab (Y-muHMFG1), PROVENGE® (sipuleucel-T),sargaramostim, sizofilan, teceleukin, THERACYS® (BacillusCalmette-Guerin), ubenimex, VIRULIZIN® (immunotherapeutic, LorusPharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF-10(Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin), ZADAXIN®(thymalfasin), ZENAPAX® (daclizumab), ZEVALIN®. (90Y-Ibritumomabtiuxetan) and the like; ofatumumab; biological response modifiers agentsincluding krestin, lentinan, sizofuran, picibanil PF-3512676 (CpG-8954),ubenimex and the like; pyrimidine analogs include cytarabine (ara C orArabinoside C), cytosine arabinoside, doxifluridine, FLUDARA®(fludarabine), 5-FU (5-fluorouracil), floxuridine, GEMZAR®(gemcitabine), TOMUDEX® (ratitrexed), TROXATYL® (triacetyluridinetroxacitabine) and the like; purine analogs including LANVIS®(thioguanine) and PURI-NETHOL® (mercaptopurine); antimitotic agentsincluding batabulin, epothilone D (KOS-862),N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,ixabepilone (BMS 247550), paclitaxel, TAXOTERE® (docetaxel), PNU100940(109881), patupilone, XRP-9881 (larotaxel), vinflunine, ZK-EPO(synthetic epothilone) and the like; and other chemotherapeutic agentssuch as ABRAXANE® (ABI-007), ABT-100 (farnesyl transferase inhibitor),ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® or MEVACOR® (lovastatin),AMPLIGE®. (poly I:poly C12U, a synthetic RNA), APTOSYN® (exisulind),AREDIA® (pamidronic acid), arglabin, L-asparaginase, atamestane(1-methyl-3,17-dione-androsta-1,4-diene), AVAGE® (tazarotene), AVE-8062(combreastatin derivative) BEC2 (mitumomab), cachectin or cachexin(tumor necrosis factor), canvaxin (vaccine), CEAVAC® (cancer vaccine),CELEUK® (celmoleukin), CEPLENE® (histamine dihydrochloride), CERVARIX®(human papillomavirus vaccine), CHOP® (C: CYTOXAN® (cyclophosphamide);H: ADRIAMYCIN® (hydroxydoxorubicin); O: Vincristine) (ONCOVIN®; P:prednisone), CYPAT® (cyproterone acetate), combrestatin A4P, DAB(389)EGF(catalytic and translocation domains of diphtheria toxin fused via aHis-Ala linker to human epidermal growth factor) or TransMID-107R®(diphtheria toxins), dacarbazine, dactinomycin,5,6-dimethylxanthenone-4-acetic acid (DMXAA), eniluracil, EVIZON™(squalamine lactate), DIMERICINE® (T4N5 liposome lotion),discodermolide, DX-8951f (exatecan mesylate), enzastaurin, EP0906(epithilone B), GARDASIL® (quadrivalent human papillomavirus (Types 6,11, 16, 18) recombinant vaccine), GASTRIMMUNE®, GENASENSE®, GMK(ganglioside conjugate vaccine), GVAX® (prostate cancer vaccine),halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101,IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonasexotoxin, interferon-.alpha., interferon-.gamma., JUNOVAN® or MEPACT®(mifamurtide), lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine(hexadecylphosphocholine), NEOVASTAT® (AE-941), NEUTREXIN® (trimetrexateglucuronate), NIPENT® (pentostatin), ONCONASE® (a ribonuclease enzyme),ONCOPHAGE® (melanoma vaccine treatment), ONCOVAX® (IL-2 Vaccine),ORATHECIN® (rubitecan), OSIDEM® (antibody-based cell drug), OVAREX® MAb(murine monoclonal antibody), paclitaxel, PANDIMEX® (aglycone saponinsfrom ginseng comprising 20(S)protopanaxadiol (aPPD) and20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigationalcancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol,procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID®(lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide),SORIATANE® (acitretin), staurosporine (Streptomyces staurospores),talabostat (PT100), TARGRETIN® (bexarotene), TAXOPREXIN®(DHA-paclitaxel), TELCYTA® (canfosfamide, TLK286), temilifene, TEMODAR®(temozolomide), tesmilifene, thalidomide, THERATOPE® (STn-KLH), thymitaq(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazolinedihydrochloride), TNFERADE® (adenovector: DNA carrier containing thegene for tumor necrosis factor-.alpha.), TRACLEER® or ZAVESCA®(bosentan), tretinoin (Retin-A), tetrandrine, TRISENOX®. (arsenictrioxide), VIRULIZIN®, ukrain (derivative of alkaloids from the greatercelandine plant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN®(motexafin gadolinium), XINLAY® (atrasentan), XYOTAX® (paclitaxelpoliglumex), YONDELIS® (trabectedin), ZD-6126, ZINECARD® (dexrazoxane),ZOMETA® (zolendronic acid), crizotinib, zorubicin and the like.

In another preferred embodiment, the therapeutic comprises a compoundthat binds to desmoglein-2; preferably a compound that binds to DSG2 andopens up tight junctions.

In other embodiments, the therapeutic comprises radioactiveparticles/radiation therapy. Any suitable radioactive therapy orparticle can be used as deemed appropriate by an attending physician,including but not limited to cobalt-60, iodine-131, iridium-192,strontium-89, samarium 153, rhenium-186 and lead-212.

In a preferred embodiment, the therapeutic is an anti-tumor therapeuticand comprises a chemotherapeutic or anti-tumor monoclonal antibody asdescribed herein. In a further preferred embodiment, the anti-tumortherapeutic comprises an antibody selected from the group consisting oftrastuzumab, cetumiximab, petuzumab, apomab, conatumumab, lexatumumab,bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab,edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab,epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomabdaclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.Non-limiting examples of useful anti-tumor mAb and their specific usesare listed in Table 1, and as further described in Campoli, M., et al.,Principles & Practice of Oncology 23(1&2):1-19 (2009), incorporatedherein by reference.

TABLE 1 Tumor-Antigen Specific mAbs for Cancer Treatment AntibodyIsotype Target Disease Indication SGN-75 humanized CD70 solid tumors,including renal cell cancer, IgG1 CD70 + hematologic malignanciesTrastuzumab humanized HER2/neu HER2/neu(+) breast cancer* IgG1 CetuximabChimeric IgG1 EGFR EGFR(+) colon cancer* Panitumumab Fully human EGFREGFR(+) colon cancer* IgG2 Matuzumab Humanized EGFR non-squamousnon-small cell lung cancer IgG1 (NSCLC), head and neck squamous cellcarcinoma (HNSCC), breast and pancreatic cancer, colon cancer (CC)Pertuzumab Humanized EGFR NSCLC, HNSCC, CC, breast and ovarian cancerIgG1 Ipilimumab (MDX- Humanized CTLA-4 NSCLC, RCC, metastatic melanoma010) IgG1 Tremelinnumab (CP- Humanized CTLA-4 NSCLC, RCC, metastaticmelanoma 675, 206) IgG1 Sibrotuzumab Humanized FAP** NSCLC, CC IgG1DR-4-specific Humanized TRAIL NSCLC, CC, ovarian cancer, multiplemapatumumab (TRM- IgG1 myeloma, 1, HGS-ETR1) DR-5-specific HumanizedTRAIL solid tumors lexatumumab (HGS- IgG1 ETR2, TRA-8) CantuzumabHumanized CanAg*** CC, pancreatic cancer mertansine IgG1- maytansinoidBevacizumab humanized vascular colon cancer*, non-squamous non-smallcell (Avastatin) IgG1 endothelial lung cancer (NSCLC)*, metastaticbreast growth cancer* factor (VEGF)

The monoclonal antibody therapeutics can be any type of monoclonalantibody, including but not limited to standard monoclonal antibodies,humanized monoclonals, fully human antibodies generated from mice orother sources, chimeric monoclonals, and fragments thereof. “Humanizedmonoclonal antibodies” refers to monoclonal antibodies derived from anon-human monoclonal antibody, such as a mouse monoclonal antibody.Alternatively, humanized monoclonal antibodies can be derived fromchimeric antibodies that retain, or substantially retain, theantigen-binding properties of the parental, non-human, monoclonalantibodies but which exhibit diminished immunogenicity as compared tothe parental monoclonal antibody when administered to humans. Forexample, chimeric monoclonal antibodies can comprise human and murineantibody fragments, generally human constant and mouse variable regions.Humanized monoclonal antibodies can be prepared using a variety ofmethods known in the art, including but not limited to (1) graftingcomplementarity determining regions from a non-human monoclonal antibodyonto a human framework and constant region (“humanizing”), and (2)transplanting the non-human monoclonal antibody variable domains, but“cloaking” them with a human-like surface by replacement of surfaceresidues (“veneering”). These methods are disclosed, for example, in,e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc.Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv.Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536(1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol.31(3):169-217 (1994); and Kettleborough, C. A. et al., Protein Eng.4(7):773-83 (1991). Monoclonal antibodies can be fragmented usingconventional techniques, and the fragments screened for utility in thesame manner as for whole antibodies. For example, F(ab′)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Fab fragments can be obtained by treating an IgG antibodywith papain; F(ab′) fragments can be obtained with pepsin digestion ofIgG antibody. A F(ab′) fragment also can be produced by binding Fab′described below via a thioether bond or a disulfide bond. A Fab′fragment is an antibody fragment obtained by cutting a disulfide bond ofthe hinge region of the F(ab′)2. A Fab′ fragment can be obtained bytreating a F(ab′)2 fragment with a reducing agent, such asdithiothreitol. Antibody fragment peptides can also be generated byexpression of nucleic acids encoding such peptides in recombinant cells(see, e.g., Evans et al., J. Immunol. Meth. 184: 123-38 (1995)). Forexample, a chimeric gene encoding a portion of a F(ab′)2 fragment caninclude DNA sequences encoding the CH1 domain and hinge region of the Hchain, followed by a translational stop codon to yield such a truncatedantibody fragment molecule. Non-limiting examples of monoclonal antibodyfragments include (i) a Fab fragment, a monovalent fragment consistingessentially of the VL, VH, CL and CH I domains; (ii) F(ab)₂ and F(ab′)2fragments, bivalent fragments comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consistingessentially of the VH and CH1 domains; (iv) a Fv fragment consistingessentially of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsessentially of a VH domain; and (vi) one or more isolated CDRs or afunctional paratope.

In one preferred embodiment that can be combined with any embodiment orcombination of embodiments of the invention, the disorder comprises aHer-2 positive tumor, and the method comprises co-administering theAdB-2/3 fiber multimer together with suitable monoclonal antibodytherapy, alone or in combination with a chemotherapeutic, radiation, orcombinations thereof. In a further preferred embodiment, the monoclonalantibody is trastuzumab. In a further preferred embodiment that can becombined with any of these embodiments, the Her-2 positive tumor isselected from the group consisting of a breast tumor, a gastric tumor, acolon tumor, and an ovarian tumor, In a most preferred embodiment, theAdB-2/3 fiber multimer comprises an Ad3 PtDd, JO-1 multimers (SEQ IDNO:20), or functional equivalents thereof. As shown in the examples thatfollow, AdB-2/3 fiber multimer co-administration with trastuzumab leadsto improved trastuzumab access to Her-2 receptors in a breast tumormodel, resulting in greatly improved trastuzumab therapeutic efficacy.In a further preferred embodiment, the method is carried out on patientswho have not responded adequately to trastuzumab, such as by lack oftumor remission, by tumor relapse, or by development of resistance totrastuzumab. The methods of these embodiments can also be used to helpreduce the dosage of trastuzumab required to obtain therapeuticefficacy, and can thus serve to limit side effects (such astrastuzumab-associated cardiotoxicity).

In another preferred embodiment that can be combined with any embodimentor combination of embodiments of the invention, the disorder comprisesan EGFR-positive tumor, and the method comprises co-administering theAdB-2/3 fiber multimer together with suitable monoclonal antibodytherapy, alone or in combination with a chemotherapeutic, radiation, orcombinations thereof. In a further preferred embodiment, the monoclonalantibody is cetuximab. In a further preferred embodiment that can becombined with any of these embodiments, the EGFR-positive tumor isselected from the group consisting of a lung tumor, a colon tumor, abreast tumor, a rectal tumor, a head and neck tumor, and a pancreatictumor. In a most preferred embodiment, the AdB-2/3 fiber multimercomprises an Ad3 PtDd, JO-1 multimers (SEQ ID NO:20), or functionalequivalents thereof. As shown in the examples that follow, AdB-2/3 fibermultimer co-administration with cetuximab leads to improved cetuximabaccess to EGFR receptors in a lung tumor model, resulting in greatlyimproved cetuximab therapeutic efficacy. In a further preferredembodiment, the method is carried out on patients who have not respondedadequately to cetuximab, such as by lack of tumor remission, by tumorrelapse, or by development of resistance to cetuximab. The methods ofthese embodiments can also be used to help reduce the dosage ofcetuximab required to obtain therapeutic efficacy, and can thus serve tolimit side effects (such as acne-like rashes that often occur duringcetuximab therapy).

In one preferred embodiment that can be combined with any embodiment orcombination of embodiments of the invention, the disorder comprises anepithelial tumor, and the method comprises co-administering the AdB-2/3fiber multimer together with a vascular endothelial growth factor (VEGF)inhibitor, alone or in combination with other chemotherapeutic,radiation, or combinations thereof. Any suitable VEGF inhibitor can beused, including but not limited to bevacizumab.

In a further embodiment that can be combined with any embodiment orcombination of embodiments herein, the methods involving solid tumorsfurther comprise administering a compound capable of degrading tumorstroma proteins. As shown in the examples that follow, such an approach(combination of compound to degrade tumor stroma protein with JO-1multimers (SEQ ID NO:20) and trastuzumab) resulted in complete tumoreradication in a breast cancer model. Any suitable compound fordegrading tumor stroma proteins can be used, including but not limitedto relaxin, collagenase, trypsin, dispase, MMP (metalloproteinase)-1,and MMP8. Delivery of such compounds can be by any suitable mechanism,including gene therapy, separate administration with the AdB-2/3 fibermultimer and the therapeutic, or administration as a conjugate with theAdB-2/3 fiber or therapeutic.

In a further embodiment that can be combined with any embodiment orcombination of embodiments herein, the methods further compriseadministering the AdB-2/3 multimer in combination with other junctionopeners. As used herein, a “junction opener” is a compound capable oftransiently opening intercellular junctions. Any suitable junctionopeners can be used. In one non-limiting embodiment, the junction openercomprises Zona occludens toxin (Zot), a Vibrio cholerae (V.cholerae)-produced toxin that possess the ability to reversibly alterintestinal epithelial junctions, allowing the passage of macromoleculesthrough mucosal barriers (Fasano et al. (1991) Proc Natl Acad Sci USA88: 5242-5246)]. A Zot-derived hexapeptide (AT-1001) has beendeveloped,) In another embodiment, Clostridium perfringens enterotoxinremoves claudins-3 and -4 from the tight junctions to facilitatebacterial invasion (Sonoda N, et al. (1999)] Cell Biol 147: 195-204.].In a further embodiment, oncoproteins encoded by human Ad, HPV, HTLV-1can transiently open epithelial junctions by mislocalizing the junctionprotein ZO-1 (Latorre I J, et al. (2005) J Cell Sci 118: 4283-4293). Inother embodiments, several human viruses engage tight junction or othercell junction molecules to achieve entry into epithelial cells. Amongthese viruses are hepatitis C virus (Evans M J, et al. (2007) Nature446: 801-805), reovirus (Barton E S, et al. (2001) Cell 104: 441-451),and herpes simplex virus (Geraghty R J, et al. (1998) Science 280:1618-1620).

In another embodiment, the therapeutic is an inhaled therapeutic. Anysuitable inhaled therapeutic can be used in the methods of theinvention. In various non-limiting embodiments, the inhaled therapeuticis selected from the group consisting of corticosteroids,bronchodilators, beta agonists, anticholinergics, albuterol (PROVENTIL®;VENOLIN®; ACCUNEB®; PROAIR®), levalbuterol) (XOPENEX®), pirbutrol(MAXAIR®), ipratropium bromide (ATROVENT®), beclomethasone, budesonide,flunisolide (AEROBID®), fluticasone, triamcinolone acetonide,fluticasone (a corticosteroid) and salmeterol (ADVAIR®), formotorol (along-acting, beta-agonist bronchodilator) and budesonide (acorticosteroid) (SYMICORT®), albuterol (a beta agonist) and ipratropium(COMBIVENT®; an anticholinergic) (budesonide (PULMICORT RESPULES®), andtiopropium (SPIRIVA®; an anticholinergic bronchodilator).

In another embodiment, the compound comprises a diagnostic or imagingagent. The methods of the invention have broad application for deliveryof any diagnostic, imaging agent, or other compound to epithelial tissuecomprising intercellular junctions where access to a target of interestcan be limited. In various non-limiting embodiments, the imaging agentscan include any chemical compound that can produce a detectable signal,either directly or indirectly. Many such imaging agents are known tothose of skill in the art. Examples of imaging agents suitable for usein the disclosed methods and compositions are radioactive isotopes,fluorescent molecules, magnetic particles (including nanoparticles),metal particles (including nanoparticles), phosphorescent molecules,enzymes, antibodies, ligands, and combinations thereof, while diagnosticagents may comprise a compound that is a diagnostic marker for aparticular epithelial disorder bound to the such an imaging agent.Methods for detecting and measuring signals generated by imaging agentsare also known to those of skill in the art. For example, radioactiveisotopes can be detected by scintillation counting or directvisualization; fluorescent molecules can be detected with fluorescentspectrophotometers; phosphorescent molecules can be detected with aspectrophotometer or directly visualized with a camera; enzymes can bedetected by detection or visualization of the product of a reactioncatalyzed by the enzyme; antibodies can be detected by detecting asecondary detection label coupled to the antibody. In one preferredembodiment, the imaging agent and/or diagnostic is one that can be usedto detect a tumor, whether by direct tumor binding, or by coupling ofthe imaging or diagnostic agent with a compound that can bind the tumor.

In one example, the imaging agents can comprise a fluorescent imagingagent, while diagnostic agents may comprise a compound that is adiagnostic marker for a particular epithelial disorder bound to thefluorescent imaging agent. A fluorescent imaging agent is any chemicalmoiety that has a detectable fluorescence signal. This imaging agent canbe used alone or in combination with other imaging agents. Examples ofsuitable fluorescent agents that can be used in the compositions andmethods disclosed herein include, but are not limited to, fluorescein(FITC), 5-carboxyfluorescein-N-hydroxysuccinimide ester,5,6-carboxymethyl fluorescein, nitrobenz-2-oxa-1,3-diazol-4-yl(NBD),fluorescamine, OPA, NDA, indocyanine green dye, the cyanine dyes (e.g.,Cy3, Cy3.5, Cy5, Cy5.5 and Cy7),4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine,acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate,N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BODIPY, BrilliantYellow, coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumaran 151), cyanosine,4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosin, eosinisothiocyanate, erythrosin B, erythrosine, isothiocyanate, ethidiumbromide, ethidium, 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate, IR144, IR1446, Malachite Green isothiocyanate,4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene,pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4(Cibacron[R] Brilliant Red 3B-A), 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), 5,6-tetramethyl rhodamine, rhodamine B, rhodamine 123,rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid,coumarin-6, and the like, including combinations thereof. Thesefluorescent imaging moieties can be obtained from a variety ofcommercial sources, including Molecular Probes, Eugene, Oreg. andResearch Organics, Cleveland, Ohio, or can be synthesized by those ofordinary skill in the art.

In another example, the imaging agents can comprise a Magnetic ResonanceImaging (MRI) agent, while diagnostic agents may comprise a compoundthat is a diagnostic marker for a particular epithelial disorder boundto the MRI agent. A MRI agent is any chemical moiety that has adetectable magnetic resonance signal or that can influence (e.g.,increase or shift) the magnetic resonance signal of another agent. Thistype of imaging agent can be used alone or in combination with otherimaging agent. In still another example, a gadolinium-based MRI agentcan serve as an imaging agent. An example of a suitable MRI agent thatcan be incorporated into the disclosed imaging agents ispara-amino-benzyl diethylenetriaminepentaacetic acid (p-NH₂-Bz-DTPA,Compound 7), a conjugable form of diethylenetriaminepentaacetic acid(DTPA), which is known to strongly bind gadolinium and is approved forclinical use as a magnetic resonance contrast agent. Incorporation of anMRI agent on a large macromolecule such as a dendrimeric substrate asdisclosed herein can allow large T1 relaxation (high contrast) andmultiple copies of agent on a single molecule, which can increasesignal. By combining an MRI imaging agent and, for example, afluorescent imaging agent, the resulting agent can be detected, imaged,and followed in real-time via MR I. Other imaging agents include PETagents that can be prepared by incorporating an 18F or a chelator for64Cu or 68Ga. Also, addition of a radionuclide can be used to facilitateSPECT imaging or delivery of a radiation dose, while diagnostic agentsmay comprise a compound that is a diagnostic marker for a particularepithelial disorder bound to the PET agent.

In some embodiments, the diagnostic agent is a diagnostic imaging agent,including but not limited to position emission tomography (PET) agents,computerized tomography (CT) agents, magnetic resonance imaging (MRI)agents, nuclear magnetic imaging agents (NMI), fluoroscopy agents andultrasound contrast agents. Such diagnostic agents include radioisotopesof such elements as iodine (I), including ¹²³I, ¹²⁵I, ¹³¹I etc., barium(Ba), gadolinium (Gd), technetium (Tc), including ⁹⁹Tc, phosphorus (P),including ³¹P, iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr),including ⁵¹Cr, carbon (C), including ¹⁴C, or the like, fluorescentlylabeled compounds, or their complexes, chelates, adducts and conjugates.Any suitable PET agents can be used, including but not limited tocarbon-11, nitrogen-13, oxygen-15, fluorine-18, 11C-metomidate, andglucose analogues thereof, including but not limited to fludeoxyglucose(a glucose analog labeled with fluorine-18.

In other embodiments, the diagnostic agent is a marker gene that encodeproteins that are readily detectable when expressed in a cell(including, but not limited to, beta-galactosidase, green fluorescentprotein, luciferase, and the like) and labeled nucleic acid probes(e.g., radiolabeled or fluorescently labeled probes). In someembodiments, covalent conjugation of diagnostics agents to the AdB-2/3multimers provided herein is achieved according to a variety ofconjugation processes. In other embodiments, the diagnostic agent isnon-covalently associated with AdB-2/3 multimers provided

In a further aspect, the present invention provides methods forimproving delivery of a substance to an epithelial tissue, comprisingcontacting the epithelial tissue with (a) one or more compound to bedelivered to the epithelial tissue; and (b) an amount of AdB-2/3 fibermultimer, or functional equivalent thereof, sufficient to enhancedelivery of the one or more compounds to the epithelial tissue. In thisaspect, the compounds may be any suitable compound such as thosedescribed in detail above. In a preferred embodiment, the one or morecompounds comprise an imaging agent. In a further preferred embodimentthe epithelial tissue comprises a solid tumor, including any of thosedisclosed in the present application. In various non-limitingembodiments, the solid tumor is selected from the group consisting ofbreast tumors, lung tumors, colon tumors, rectal tumors, stomach tumors,prostate tumors, ovarian tumors, uterine tumors, skin tumors, endocrinetumors, cervical tumors, kidney tumors, melanomas, pancreatic tumors,liver tumors, brain tumors, head and neck tumors, nasopharyngeal tumors,gastric tumors, squamous cell carcinomas, adenocarcinomas, bladdertumors, and esophageal tumors.

In a still further aspect, the present invention provides methods forimproving delivery of a substance cell or tissue expressing desmoglein 2(DSG2), comprising contacting the cell or tissue expressing DSG2 with(a) one or more compound to be delivered to the cell or tissue; and (b)an amount of AdB-2/3 fiber multimer, or functional equivalent thereof,sufficient to enhance delivery of the one or more compounds to thetissue. Exemplary tissue types expressing DSG2 include, but are notlimited to epithelial cells/tissue (such as those disclosed herein),human platelets and granulocytes. As shown in the examples that follow,DSG2 also acts as receptor in non-polarized cells. Thus, these methodsfind application not only in epithelial cells and tissue, but also arerelevant, for example, in AdB-2/3 pathogenesis and the intravascularapplication of AdB-2/3 vectors for gene therapy purposes.

In a still further aspect, the present invention provides methods forinducing an epithelial to mesenchymal transition (EMT) in a tissue,comprising contacting the epithelial tissue with an amount of AdB-2/3fiber multimer, or functional equivalent thereof, sufficient to induceEMT. EMT is a cellular transdifferentiation program where epithelialcells lose characteristics such as intercellular junctions and gainproperties of mesenchymal cells. EMT is characterized by increasedexpression of mesenchymal markers, increased expression of extracellularmatrix compounds, decreased expression of epithelial markers, alteredlocation of transcription factors, and activation of kinases, anddisassociation of intercellular junctions.

In each of these further aspects, any embodiment of the compounds andAdB-2/3 fiber multimers described herein can be used. In onenon-limiting embodiment, the AdB-2/3 fiber multimer is selected from thegroup consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, anAd11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer,combinations thereof, and functional equivalents thereof. In anotherembodiment, the AdB-2/3 fiber multimer is an Ad3 fiber multimer, or afunctional equivalent thereof. In another embodiment, the AdB-2/3 fibermultimer is selected from the group consisting of AdB-2/3 virions,AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd), recombinantAdB-2/3 fiber multimers, and functional equivalents thereof. In variouspreferred embodiments, the AdB-2/3 fiber multimer comprises an Ad3 PtDdor a junction opener 1 (JO-1) (SEQ ID NO:20) multimer (such as a dimer),or functional equivalent thereof. In a further preferred embodiment thatcan be combined with each of these embodiments, the AdB-2/3 fibermultimer is a dimer. While not being bound by any specific mechanism ofaction, it is believed that these further aspects each take advantage ofthe AdB-2/3 fiber multimer serving to disrupt intercellular junctions.

In all of the aspects and embodiments of the methods of the invention,the therapeutic, diagnostic, and/or imaging agent can be administeredtogether with the AdB-2/3 multimer or may be administered together. Inone embodiment, the therapeutic and AdB-2/3 multimer are attached, viaany suitable covalent or non-covalent binding. In one non-limitingembodiment, an AbB-2/3 multimer can attached to a toxin or other drug tokill solid tumor cells.

The AdB-2/3 fiber multimer and/or therapeutic can be administered in anyway deemed suitable by an attending physician, depending on whether alocal or systemic mode of administration is most appropriate for thecondition being treated. As used herein, the terms “systemic delivery”and “systemic administration” are intended to include, but are notlimited to, oral and parenteral routes including intramuscular (IM),subcutaneous, intravenous (IV), intra-arterial, inhalational,sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, andother routes of administration that effectively result in dispersementof the delivered agent to a single or multiple sites of intendedtherapeutic action. Preferred routes of systemic delivery for thepresent compositions include intravenous, intramuscular, subcutaneous,and inhalational. In one preferred embodiment, intravenousadministration is used, such as for treatment of disseminated tumors(and for monoclonal antibody delivery). In another embodiment, oraldelivery may be preferred, for example, for treating gastrointestinal(GI) epithelial disorders. In another embodiment, nasal or aerosoldelivery may be preferred for delivery to the lungs, such as for lungepithelial disorders.

The AdB-2/3 fiber multimer may be introduced in association with anothermolecule, such as a lipid or liposome to protect the polypeptides fromenzymatic degradation. For example, the covalent attachment of polymers,especially polyethylene glycol (PEG), has been used to protect certainproteins from enzymatic hydrolysis in the body and thus prolonghalf-life.

The AdB-2/3 fiber multimer and/or therapeutic may be systemicallyadministered on a periodic basis at intervals determined to maintain adesired level of therapeutic effect. For example, administration byintravenous injection may be once per day, once per week, every two tofour weeks or at less frequent intervals. The dosage regimen will bedetermined by the physician considering various factors that mayinfluence the action of the combination of agents. These factors willinclude the extent of progress of the condition being treated, thepatient's age, sex and weight, and other clinical factors. The dosagefor AdB-2/3 fiber multimer and/or therapeutic will vary as a function ofthe multimer and/or therapeutic being administered, as well as thepresence and nature of any drug delivery vehicle (e.g., a sustainedrelease delivery vehicle). In addition, the dosage quantity may beadjusted to account for variation in the frequency of administration andthe pharmacokinetic behavior of the delivered agent(s). Dosage ranges ofAdB-2/3 fiber multimers will generally range between 0.01 and 250 mg/kg,preferably between 0.1 and 10 mg/kg, and more preferably between 0.10 to0.5 mg/kg. Dosages of approved therapeutics are readily identifiable bymedical practitioners. The therapeutic may also be able to beadministered at a reduced dose due to enhanced penetration intoepithelial tissues, such as cancers.

The AdB-2/3 fiber multimer may be administered to the subject before,simultaneously, or after administration of the therapeutic. In apreferred embodiment, administration of the therapeutic and the AdB-2/3fiber multimer are carried out at the same time. The timing ofadministrations of the therapeutic relative to the AdB-2/3 fibermultimer can be varied to achieve the greatest therapeutic effect.Preferably, the therapeutic is administered at a time to ensure itscontact with the transient opening of the intercellular junction causedby AdB-2/3 fiber multimer binding to DSG2. For example, the therapeuticcan be administered prior to, simultaneously with, after eachadministration of the AdB-2/3 fiber multimer. In other preferredembodiments, the therapeutic can be administered after theadministration of the AdB-2/3 fiber multimer, for example up to 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours,18 hours, 24 hours, 30 hours, 36 hours, 40 hours, 42 hours, 48 hours, 54hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, oreven up to 96 hours after the administration of the AdB-2/3 fiber.multimer.

In another aspect, the present invention provides methods for treating adisorder associated with epithelial tissue, comprising administering toa subject in need thereof an amount of AdB-2/3 fiber multimer, orfunctional equivalent thereof, sufficient to treat the disorder. In thisembodiment, no other therapeutic is delivered. In non-limitingembodiments, the monotherapy is used to treat a disorder selected fromthe group consisting of an AdB-2/3 viral infection, a solid tumor, or adisorder that can be treated using an AdB-2/3-based gene deliveryvector. For example, in treating solid tumors, the method comprisesimproving access of immune system cells to the site of the disorder,such as by penetration (such as intratumoral penetration of pre-existingnatural killer cells, T-cells or dendritic cells). The method can alsobe used to treat any of the disorders associated with epithelial cellsdiscussed above that can benefit from improved access of cells of theimmune system to the target epithelial cells. In one preferredembodiment, the disorder is a solid tumor, and the method comprisesimproving immune system attack of the tumor. The method can be used withany of the solid tumors discussed above. All embodiments andcombinations of embodiments of the first aspect of the invention can beused in this second aspect as well, unless the context clearly dictatesotherwise.

Thus, in various embodiments of this second aspect, the AdB-2/3 fibermultimer is selected from the group consisting of an Ad3 fiber multimer,an Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer,and an Ad14a fiber multimer. Similarly, exemplary constructs comprisingone or more AdB-2/3 fiber multimers (or chimeras/functional equivalentsthereof) for use in this aspect of the invention include, but are notlimited to, AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedralparticles (PtDd) (subviral dodecahedral particles produced by AdB-2/3during their replication), recombinant AdB-2/3 fiber multimers(including but not limited to those disclosed in any embodiment orcombination of embodiments below, such as a JO-1 multimer, such as aJO-1 dimer), and functional equivalents thereof. In a preferredembodiment, the one or more AdB-2/3 fiber multimers comprise or consistof an AdB-2/3 PtDd, such as an Ad3 PtDd. In another preferredembodiment, the one or more AdB-2/3 fiber multimers comprise or consistof any embodiment or combination of embodiments of the compositions ofthe invention described below, such as a JO-1 dimer. Further, themethods of this aspect of the invention can be carried out using anyAdB-2/3 fiber multimer capable of binding to DSG2 and triggeringtransient DSG2-mediated opening of intercellular junctions. Thus,non-naturally occurring modifications (deletions, additions,substitutions, chimeras of different Ad serotype fiber proteins anddomains thereof, etc.) to the AdB-2/3 fiber multimers disclosed hereinare within the scope of the present invention, so long as they functionto bind DSG2 and trigger DSG2-mediated opening of intercellularjunctions. Based on the teachings herein for testing binding to DSG2 andfor assessing DSG2-mediated opening of intercellular junctions, it iswell within the level of skill in the art to identify such functionalequivalents of the AdB-2/3 fiber multimers.

In another aspect, the present invention provides recombinant AdB-2/3fiber polypeptides, comprising: (a) one or more AdB-2/3 fiberpolypeptide shaft domains, or functional equivalents thereof; (b) anAdB-2/3 fiber polypeptide knob domain, or functional equivalent thereof,operatively linked to and located C-terminal to the one or more AdB-2/3fiber protein shaft domains; and (c) one or more non-AdB-2/3-deriveddimerization domains operatively linked to and located N-terminal to theone or more AdB-2/3 fiber polypeptide shaft domains.

In a preferred embodiment, the recombinant polypeptides do not include atail domain from an Ad fiber polypeptide. As is disclosed in detailbelow, the inventors have localized the required sites for fiberpolypeptide multimerization and binding to DSG2 to the shaft and knobdomains of the AdB-2/3 proteins. The polypeptides of this aspect of theinvention can thus be used, for example, to form AdB-2/3 fiber multimersfor use in the various methods of the invention discussed above. In thisaspect, the recombinant polypeptides can include shaft and knob domainsfrom any AdB-2/3 virus, or any mutants (substitutions, additions,deletions, chimeras, etc.) to such shaft and knob domains that retain orimprove binding affinity to DSG2, and are capable of forming multimers(such as dimers) via the dimerization domain (functional equivalents).It is well within the level of skill in the art to generate suchmutants, and testing of such mutants for DSG2 binding and resultingsuitability for use in the recombinant polypeptides of the invention isalso within the level of skill in the art based on the teachings herein.For example, surface plasmon resonance (SPR) studies using sensorscontaining immobilized recombinant DSG2 can be used to determine ifrecombinant polypeptides being assessed bind to DSG2, combined with DSG2competition studies. In one non-limiting embodiment, the polypeptide maybe modified or mutated to reduce its immunogenicity by changing itsamino acid sequence using techniques well known to those skilled in theart. Further exemplary studies, such as loss and gain of functionanalyses, are described in detail in Example 1.

As used throughout the present application, the term “polypeptide” isused in its broadest sense to refer to a sequence of subunit aminoacids. The polypeptides of the invention may comprise L-amino acids,D-amino acids (which are resistant to L-amino acid-specific proteases invivo), or a combination of D- and L-amino acids. The polypeptidesdescribed herein may be chemically synthesized or recombinantlyexpressed. The polypeptides may be linked to other compounds to promotean increased half-life in vivo, such as by PEGylation, HESylation,PASylation, glycosylation, or may be produced as an Fc-fusion or indeimmunized variants. Such linkage can be covalent or non-covalent as isunderstood by those of skill in the art.

As used herein, the term “operatively linked” refers to an arrangementof elements wherein the domains are configured so that they function asa unit for their intended purpose. The term does not require that thedomains are immediately adjacent on the polypeptide, as spacer/linkersequences may be present between the domains, the lengths of which canbe quite variable. In one non-limiting embodiment, the spacer lengthbetween any two domains of the recombinant AdB-2/3 fiber polypeptidescan be between about 0 amino acids and about 20 amino acids. In variousother non-limiting embodiments, the spacer length can be 0-20, 0-19,0-18, 0-17, 0-16, 0-15, 0-14, 0-13, 0-12, 0-11, 0-10, 0-9, 0-8, 0-7,0-6, 0-5, 0-4, 0-3, 0-2, 0-1, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14,1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20,2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8,2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13,3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17,4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20,5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8,5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11,6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14,8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14,9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14,10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14,11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13,13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18,14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19,16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, 19-20, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acidsin length.

As used herein, “recombinant polypeptide” means a non-naturallyoccurring protein product, wherein the domains of the recombinantpolypeptide are derived from one or more other proteins or artificiallyderived sequences. For example, each domain can be derived from adifferent naturally occurring protein sequence (ex: shaft sequence fromone AdB-2/3 serotype; knob domain from a different AdB-2/3 serotype;etc.). The recombinant polypeptide may be constructed by a variety ofmechanisms including, but not limited to, standard DNA manipulationtechniques and chemical assembly via subunit parts of the recombinantpolypeptide. The chemical assembly may lead to an equivalent form as themolecular genetic form or alternative associations with equivalentfunction. In a preferred embodiment, the recombinant polypeptide isproduced by standard recombinant DNA techniques. Techniques for suchrecombinant production and isolation of the recombinant polypeptides ofthe invention are well within the level of skill in the art based on theteaching herein.

In one embodiment, each shaft domain is selected from the groupconsisting of an Ad3 shaft domain, an Ad7 shaft domain, an Ad11 shaftdomain, an Ad 14 shaft domain, an Ad14a shaft domain, combinationsthereof, and functional equivalents thereof. The shaft domain isrequired for fiber knob dimerization, which is required for binding toDSG2 and resulting transient opening of intercellular junctions. Thus,functional equivalents of the shaft domains of these Ad virus serotypescan be readily determined by those of skill in the art, based on theexamples provided below. For example, surface plasmon resonance (SPR)studies using sensors containing immobilized recombinant DSG2 can beused to determine if recombinant polypeptides being assessed bind toDSG2, combined with DSG2 competition studies. Further exemplary studies,such as loss and gain of function analyses, are described in detail inExample 1.

The recombinant polypeptides may comprise between 1 and 22 AdB-2/3 fiberpolypeptide shaft domains. Thus, in various embodiments to polypeptidescomprise 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13,1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-22, 2-21,2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9,2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16,3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-22,4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10,4-9, 4-8, 4-7, 4-6, 4-5, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15,5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-22, 6-21, 6-20,6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8,6-7, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12,7-11, 7-10, 7-9, 7-8, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15,8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17,9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-22, 10-21, 10-20, 10-19,10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-22, 11-21,11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-22,12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-22,13-21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-22, 14-21,14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-22, 15-21, 15-20, 15-19,15-18, 15-17, 15-16, 16-22, 16-21, 16-20, 16-19, 16-18, 16-17, 17-22,17-21, 17-20, 17-19, 17-18, 18-22, 18-21, 18-20, 18-19, 19-22, 19-21,19-20, 20-22, 20-21, 21-22, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, or 22 AdB-2/3 fiber protein shaftdomains. Where more than 1 AdB-2/3 fiber protein shaft domain ispresent, each shaft domain can be identical, or one or more copies ofthe shaft domain may differ in a single recombinant polypeptide. In apreferred embodiment, the recombinant AdB-2/3 fiber polypeptide has asingle shaft domain.

In another embodiment, one or more (or all) shaft domains in therecombinant polypeptide comprise or consist of an amino acid sequenceaccording to SEQ ID NO 11:

GVL(T/S)LKC(L/V)(T/N)PLTT(T/A)(G/S)GSLQLKVG(G/S)GLTVD(D/T)T(D/N)G(T/F/S)L(Q/K/E)ENI(G/S/K)(A/V)(T/N)TPL(V/T)K(T/S)(G/N)HSI(G/N)L(S/P)(L/I)G(A/P/N)GL(G/Q)(T/I)(D/E)(E/Q)NKLC(T/S/A)KLG(E/Q/N)GLTF(N/D)S(N/S)N(I/S)(C/I)(I/A)(D/N/L)(D/K)N(I/--)NTL.In this sequence and other variable sequences shown herein, the variableresidues are noted within parentheses, and a “−” indicates that theresidue may be absent.

In another embodiment, one or more (or all) shaft domains in therecombinant polypeptide comprise or consist of an amino acid sequenceaccording to SEQ ID NO 12:

GVLTLKCLTPLTTTGGSLQLKVGGGLT(V/I)DDTDG(T/F)L(Q/K)ENI(G/S)ATTPLVKTGHSIGL(S/P)LG(A/P)GLGT(D/N)ENKLC(T/A)KLG(E/Q)GLTFNSNNICI(D/N)DNINTL

In a still further embodiment, one or more (or all) shaft domains in therecombinant polypeptide comprise or consist of an amino acid sequenceselected from the group consisting of SEQ ID NO:1 (Ad3), SEQ ID NO:2(Ad7), SEQ ID NO:3 (Ad11), SEID NO:4 (Ad14), and SEQ ID NO:5 (Ad14(a).See FIG. 24 for an alignment of the AdB-2/3 fiber polypeptides and theirdomain structures. In one embodiment, the knob domain in the recombinantpolypeptide is selected from the group consisting of an Ad3 knob domain,an Ad7 knob domain, an Ad11 knob domain, an Ad 14 knob domain, an Ad14aknob domain, and functional equivalents thereof. The knob domain isrequired for binding to DSG2. Thus, functional equivalents of the knobdomains of these Ad virus serotypes can be readily determined by thoseof skill in the art, based on the teachings herein of various assays forassessing binding to DSG2. For example, surface plasmon resonance (SPR)studies using sensors containing immobilized recombinant DSG2 can beused to determine if recombinant polypeptides being assessed bind toDSG2, combined with DSG2 competition studies. Further exemplary studies,such as loss and gain of function analyses, are described in detail inExample 1.

In another embodiment, the knob domain comprises or consists of an aminoacid sequence according to SEQ ID NO 13:

WTG(V/P)(N/K)P(T/)(E/R)ANC(Q/I)(M/I)(M/E)(Y/A/N/D)(S/K)(S/K)(E/Q)(S/N)(N/P)D(C/S)KL(I/T)L(I/T)LVK(T/N)G(A/I)(L/I)V(T/N)(A/G)(F/Y)V(Y/T)(V/L)(I/M)G(V/A)S(N/D)(N/D/Y)(F/V)N(M/T)L(T/F)(T/K)(Y/H/N)(R/K)N(I/V)(N/S)(F/I)(T/N)(A/V)EL(F/Y)FD(S/A)(A/T)G(N/H)(L/I)L(T/P)(S/R/D)(L/S)SSLKT(P/D)L(N/E)(H/L)K(S/Y)(G/K)Q(N/T)(M/--)(A/--)(T/--)(G/--)A(I/L/D)(T/F)(N/S)A(K/R)(S/G)FMPSTTAYPF(--/V)(--/L)(N/P)(N/D/V)(N/A)(S/G)(R/T)(E/H)(N/K/--)(--/E)NYI(Y/F)G(T/Q)C(H/Y)Y(T/K)ASD(H/G)(T/A)(A/L)FP(I/L)(D/E)(I/V)(S/T)VMLN(Q/R/K)R(A/L)(I/L/P)(R/N/D)(A/D/N/S)(D/E/R)TSY(C/V)(I/M)(R/T)(I/V/F)(T/L)WS(W/L)N(T/A)G(D/L/V)APE(G/V/--)(Q/--)T(S/T)(A/Q)(T/A)TL(V/I)TSPFTF(Y/S)YIREDD.

In a further embodiment, the knob domain comprises or consists of anamino acid sequence according to SEQ ID NO:14:

WTGVNPT(E/R)ANCQ(M/I)(M/I)(D/N/A)SSESNDCKLILTLVKTGALVTAFVYVIGVSN(N/D)FNMLTT(Y/H)(R/K)NINFTAELFFDS(A/T)GNLLT(S/R)LSSLKTPLNHKSGQNMATGA(I/L)TNAK(S/G)FMPSTTAYPFN(N/D/V)NSRE(--/K)(-/E)NYIYGTC(H/Y)YTASD(H/R)TAFPIDISVMLN(Q/R)RA(I/L)(R/N)(A/D/N)(D/E)TSYCIR(I/V)TWSWNTG(D/V)APE(G/V)QTSATTLVTSPFTFYYIREDD

In a still further embodiment, the knob domain comprises or consists ofan amino acid sequence selected from the group consisting of SEQ ID NO:6(Ad3), SEQ ID NO:7 (Ad7), SEQ ID NO:8 (Ad11), SEQ ID NO:9 (Ad14), andSEQ ID NO:10 (Ad14a). See FIG. 24 for an alignment of the AdB-2/3 fiberpolypeptides and their domain structures.

As used herein a “dimerization domain” is a peptide sequence thatpromotes dimerization in the recombinant polypeptide that contains it.Any suitable non-AdB-2/3-derived dimerization domain can be used in therecombinant polypeptide of the invention, so long as it permitsdimerization of the recombinant polypeptide and thus binding to DSG2.The dimerization domain is non-AdB-2/3-derived, in that it is not anaturally occurring domain in an AdB-2/3 fiber polypeptide.

Non-limiting examples of the numerous dimerization domains known tothose of skill in the art and suitable for use in the present inventioninclude, but are not limited to peptide helices containing at least onehelix, or a structure formed by a helix, a coil and another helix, etc.,coiled coil structures, dimerization domains within, for example, manycell surface signaling receptors, Fc regions or hinge regions of anantibody, leucine zippers, the STAT protein N terminal domain, FK506binding protein, the LexA protein C-terminal domain, nuclear receptors,the FkpA N-terminal domain, orange carotenoid protein from A. maxima, M1matrix protein from influenza, neuraminidase from influenza virus, E.coli fuculose aldolase; and the like. (see, e.g., O'Shea, Science. 254:539 (1991), Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211:121-128 (1996); Klemm et al., Annu. Rev. Immunol. 16: 569-592 (1998);Klemm et al., Annu. Rev. Immunol. 16: 569-592 (1998); Ho et al., Nature.382: 822-826 (1996); and Pomeranz et al., Biochem. 37: 965 (1998)).Further examples include residues 325 to 410 in the bovinepapillomavirus E2 protein, (Dostatni, N., et al., EMBO J. 7 (1988)3807-3816; Haugen, T., et al. EMBO J 7 (1988) 4245-4253; McBride, A., etal., EMBO J. 7 (1988) 533-539; McBride, A., et al., Proc Natl Acad SciUSA 86 (1989) 510-514), Type I deiodinase (D1): DFLVIYIEEAHASDGW (SEQ IDNO: 24) or ADFL--YI-EAH-DGW (SEQ ID NO: 25); HIV-1 Capsid Protein:QGPKEPFRDYVDRFYKTLRA (SEQ ID NO: 26); leucine zipper dimerization motifof yeast GCN4: HMKQL D VEELS NYHL N VARL K VGER (SEQ ID NO: 27); leucinezipper in Escherichia coli transcriptional antiterminator protein; andBgIG: GVTQLMREMLQLIKFQFSLNYQEESLSYQRLVT (SEQ ID NO: 28). In preferredembodiments, the dimerization domain comprises one or more copies ofEVSALEK (SEQ ID NO:22) and/or KVSALKE (SEQ ID NO: 23).

It is well within the level of skill in the art to identify appropriatepeptide sequences that can serve as dimerization domains, and mutantsthereof, in the recombinant polypeptides of the present invention, basedon the teachings herein, such as those disclosed below in Example 2. Forexample, dimerization of the recombinant AdB-2/3 fiber polypeptides canbe assessed by criteria including sedimentation in sucrose gradients,resistance to trypsin proteolysis, and electrophoretic mobility inpolyacrylamide gels (Hong and Engler, Journal of Virology 70:7071-7078(1996)).

The recombinant polypeptides may comprise one or morenon-AdB-2/3-derived dimerization domains. Thus, in various embodiments,the recombinant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore non-AdB-2/3-derived dimerization domains. Where multiple domainsare present in a polypeptide, it is preferred that each dimerizationdomain is the same.

In a preferred embodiment a spacer peptide is located between thedimerization domain and the one or more shaft domains. In a furtherpreferred embodiment, the spacer peptide is a peptide with structuralflexibility. Virtually any peptide with structural flexibility can beused. As an example, the flexible peptide may comprise repetitions ofamino acid residues, such as Gly-Gly-Gly-Ser, or any other suitablerepetition of amino acid residues. In another embodiment, the hingeregion of an antibody can be used. The spacer can be any suitable lengththat maintains the ability of the recombinant polypeptide to dimerizeand to maintain binding of the recombinant polypeptide to DSG2.

In one preferred embodiment, the recombinant AdB-2/3 polypeptidecomprises:

-   -   (a) one or more shaft domains that each comprise or consist of        an Ad3 shaft domain (SEQ ID NO:1); and    -   (b) a knob domain comprises or consists of an Ad3 knob domain        (SEQ ID NO:6).

This preferred embodiment can be used with any embodiment or combinationof embodiments described herein. For example, any suitable dimerizationdomain can be used, including but not limited to one or more copies ofEVSALEK (SEQ ID NO:22) and/or KVSALKE (SEQ ID NO: 23). Similarly anysuitable spacer peptides can be used between the dimerization domain andthe shaft domain and/or between the shaft domain and the knob domain. Ina most preferred embodiment, the recombinant AdB-2/3 polypeptidecomprises or consists of JO-1 (SEQ ID NO:20), or a multimer thereof(such as a dimer).

The recombinant polypeptides may comprise further domains, such as adomain for isolation of the polypeptide and/or a detection domain, see,for example, JO-1 with a His Tag in the examples and in SEQ ID NO:21).An isolation domain can be added to facilitate purification/isolation ofthe polypeptide following, for example, recombinant polypeptideproduction. Any suitable isolation domain can be used, including but notlimited to HIS, CBP, CYD (covalent yet dissociable NorpD peptide), StrepII, FLAG, HPC (heavy chain of protein C) peptide tags, GST and MBPaffinity tags. As used herein, “detection domain” means one or moreamino acid sequence that can be detected. Any suitable detection domaincan be used, including but not limited to, inherently fluorescentproteins (e.g. Green Fluorescent Proteins and fluorescent proteins fromnonbioluminescent Anthozoa species), cofactor-requiring fluorescent orluminescent proteins (e.g. phycobiliproteins or luciferases), andepitopes recognizable by specific antibodies or other specific naturalor unnatural binding probes, including, but not limited to, dyes, enzymecofactors and engineered binding molecules, which are fluorescently orluminescently labeled.

In another embodiment, the recombinant polypeptides are in a multimericform, such as a dimer, trimer, etc. In a preferred embodiment, a JO-1(SEQ ID NO:20) multimer comprises a JO-1 dimer formed by dimerizationthrough the dimerization domains in each JO-1 homotrimer (ie: a JO-1polypeptide is a homotrimer through trimerization of the knob domain) Inmultimeric form (such as a dimer), the recombinant polypeptides compriseAdB-2/3 fiber multimers, and can be used in the various methods of theinvention discussed above. As will be understood by those of skill inthe art, such multimers may comprise multimers of identical recombinantpolypeptide of the invention, or may comprise multimers of differentrecombinant polypeptides of the invention. In one embodiment, thedimerization domains are the same in each recombinant polypeptideforming part of the multimer. In another embodiment, the dimerizationdomains are different in each recombinant polypeptide forming part ofthe multimer. In another embodiment, the shaft and/or knob domains arethe same in each recombinant polypeptide forming part of the multimer.In another embodiment, the shaft and/or knob domains are different ineach recombinant polypeptide forming part of the multimer.

AdB-2/3 fiber multimerization can be determined according to methodswell known to the practitioners in the art. For example, multimerizationof the recombinant AdB-2/3 fiber constructs can be assessed by criteriaincluding sedimentation in sucrose gradients, resistance to trypsinproteolysis, and electrophoretic mobility in polyacrylamide gels (Hongand Engler, Journal of Virology 70:7071-7078 (1996)). Regardingelectrophoretic mobility, the fiber multimer is a very stable complexand will run at a molecular weight consistent with that of a multimerwhen the sample is not boiled prior to SDS-PAGE. Upon boiling, however,the multimeric structure is disrupted and the protein subsequently runsat a size consistent with the protein monomer.

The recombinant polypeptides, or multimeric versions thereof, may bestored in solution or frozen.

In another embodiment, the recombinant polypeptides of the invention arecombined with (such as conjugated to) one or more therapeutics for adisorder associated with epithelial tissue. Such conjugates can be used,for example, in the therapeutic methods of the invention. Methods forconjugating the polypeptides of the invention to a therapeutic ofinterest, such as by covalent binding or chemical cross-linking, arewell known to those of skill in the art. Any suitable therapeutic can beused to form a conjugate according to this embodiment of the invention,including but not limited to those disclosed above, as well as tumorstroma degrading compounds, such as relaxin. In a preferred embodiment,the therapeutic is an anti-tumor therapeutic and comprises achemotherapeutic or anti-tumor monoclonal antibody as described herein.In a further preferred embodiment, the anti-tumor therapeutic comprisesan antibody selected from the group consisting of trastuzumab,cetumiximab, petuzumab, Apomab, conatumumab, lexatumumab, bevacizumab,bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab,ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab,gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab,catumaxomab, ofatumumab, and ibritumomab.

In another aspect, the present invention provides nucleic acids encodingthe polypeptide or any embodiment of the invention. The nucleic acidsmay comprise RNA or DNA, and can be prepared and isolated using standardmolecular biological techniques, based on the teachings herein. Thenucleic acids may comprise additional domains useful for promotingexpression and/or purification of the encoded protein, including but notlimited to polyA sequences, modified Kozak sequences, and sequencesencoding epitope tags, export signals, and secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In a further aspect, the present invention provides recombinantexpression vectors comprising the nucleic acid of any aspect of theinvention operatively linked to a promoter. “Recombinant expressionvector” includes vectors that operatively link a nucleic acid codingregion or gene to any promoter capable of effecting expression of thegene product. The promoter sequence used to drive expression of thedisclosed nucleic acids in a mammalian system may be constitutive(driven by any of a variety of promoters, including but not limited to,CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number ofinducible promoters including, but not limited to, tetracycline,ecdysone, steroid-responsive). The construction of expression vectorsfor use in transfecting prokaryotic cells is also well known in the art,and thus can be accomplished via standard techniques. (See, for example,Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N. J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.). The expression vector must be replicable in the host organismseither as an episome or by integration into host chromosomal DNA, andmay comprise any other components as deemed appropriate for a given use,including but not limited to selection markers such as anantibiotic-resistance gene.

In a still further aspect, the present invention provides host cellscomprising the recombinant expression vectors disclosed herein, andprogeny thereof, wherein the host cells can be either prokaryotic oreukaryotic. The cells can be transiently or stably transfected. Suchtransfection of expression vectors into prokaryotic and eukaryotic cellscan be accomplished via any technique known in the art, including butnot limited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc.New York, N. Y.). Techniques utilizing cultured cells transfected withexpression vectors to produce quantities of polypeptides are well knownin the art.

In another aspect, the present invention provides pharmaceuticalcompositions, comprising

(a) an AdB-2/3 fiber multimer, or functional equivalent thereof; and

(b) a pharmaceutically acceptable carrier.

The AdB-2/3 fiber multimer can be any such multimer as described hereinaccording to any aspect, embodiment, or combination of embodiments ofthe invention. In various preferred embodiments, the AsB-2/3 fibermultimer comprises an AdB-2/3 virion, an AdB-2/3 capsid, an AdB-2/3PtDd, or a recombinant AdB-2/3 fiber multimer of the invention, orfunctional equivalents thereof. In various other preferred embodiments,the AdB-2/3 fiber multimer is selected from the group consisting of anAd3 fiber multimer, an Ad7 fiber multimer, an Ad11 fiber multimer, anAd14 fiber multimer, an Ad14a fiber multimer, and combinations orchimeras thereof. In a preferred embodiment, the one or more AdB-2/3fiber multimers comprise or consist of an AdB-2/3 PtDd (such as Ad3PtDd). In another preferred embodiment, the one or more AdB-2/3 fibermultimers comprise or consist of any embodiment or combination ofembodiments of the compositions of the invention described herein, suchas a JO-1 (SEQ ID NO:20) dimer.

The pharmaceutical composition may further comprise one or moretherapeutic for treating a disorder associated with epithelial tissue,including but not limited to those disclosed above. In a preferredembodiment, the therapeutic is an anti-tumor therapeutic and comprises achemotherapeutic or anti-tumor monoclonal antibody as described herein.In a further preferred embodiment, the anti-tumor therapeutic comprisesan antibody selected from the group consisting of trastuzumab,cetumiximab, petuzumab, Apomab, conatumumab, lexatumumab, bevacizumab,bevacizumab, denosumab, zanolimumab, Iintuzumab, edrecolomab, rituximab,ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab,gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab,catumaxomab, ofatumumab, and ibritumomab.

The pharmaceutically acceptable carrier is non-toxic, biocompatible andis selected so as not to detrimentally affect the biological activity ofthe multimers (and any other therapeutic agents combined therewith).Exemplary pharmaceutically acceptable carriers for peptides aredescribed in U.S. Pat. No. 5,211,657 to Yamada. The compositions may beformulated into preparations in solid, semi-solid, gel, liquid orgaseous forms such as tablets, capsules, powders, granules, ointments,solutions, suppositories, inhalants, and injections, allowing for oral,parenteral, or surgical administration. Suitable carriers for parenteraldelivery via injectable, infusion, or irrigation and topical deliveryinclude distilled water, physiological phosphate-buffered saline, normalor lactated Ringer's solutions, dextrose solution, Hank's solution, orpropanediol. In addition, sterile, fixed oils may be employed as asolvent or suspending medium. For this purpose any biocompatible oil maybe employed including synthetic mono- or diglycerides. In addition,fatty acids, such as oleic acid, find use in the preparation ofinjectables. The carrier and agent may be compounded as a liquid,suspension, polymerizable or non-polymerizable gel, paste or salve. Thecarrier may also comprise a delivery vehicle to sustain (i.e., extend,delay, or regulate) the delivery of the agent(s) or to enhance thedelivery, uptake, stability, or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexample, microparticles, microspheres, nanospheres, or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels, andpolymeric micelles. Suitable hydrogel and micelle delivery systemsinclude the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexesdisclosed in International Publication No. WO 2004/009664 A2, and thePEO and PEO/cyclodextrin complexes disclosed in U.S. Publication No.2002/0019369 A1. Such hydrogels may be injected locally at the site ofintended action, or subcutaneously or intramuscularly to form asustained release depot.

For intrathecal (IT) or intracerebroventricular (ICV) delivery,appropriately sterile delivery systems (e.g., liquids; gels,suspensions, etc.) can be used to administer the compositions. For oraladministration of non-peptidergic agents, the compositions may becarried in an inert filler or diluent such as sucrose, cornstarch, orcellulose.

The compositions of the present invention may also include biocompatibleexcipients, such as dispersing or wetting agents, suspending agents,diluents, buffers, penetration enhancers, emulsifiers, binders,thickeners, flavoring agents (for oral administration). Exemplaryformulations can be parenterally administered as injectable dosages of asolution or suspension of the multimer in a physiologically acceptablediluent with a pharmaceutical carrier that can be a sterile liquid suchas water, oils, saline, glycerol, or ethanol. Additionally, auxiliarysubstances such as wetting or emulsifying agents, surfactants, pHbuffering substances and the like can be present in compositionscomprising modified polypeptides. Additional components ofpharmaceutical compositions include petroleum (such as of animal,vegetable, or synthetic origin), for example, soybean oil and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers for injectable solutions.

The pharmaceutical composition can also be administered in the form of adepot injection or implant preparation that can be formulated in such amanner as to permit a sustained or pulsatile release of the multimersand other therapeutic (if present).

The pharmaceutical composition may comprise in addition to thepolypeptide of the invention (a) a lyoprotectant; (b) a surfactant; (c)a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) apreservative and/or (g) a buffer. In some embodiments, the buffer in thepharmaceutical composition is a Tris buffer, a histidine buffer, aphosphate buffer, a citrate buffer or an acetate buffer. Thepharmaceutical composition may also include a lyoprotectant, e.g.sucrose, sorbitol or trehalose. In certain embodiments, thepharmaceutical composition includes a preservative e.g. benzalkoniumchloride, benzethonium, chlorohexidine, phenol, m-cresol, benzylalcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride.

The pharmaceutical composition can be packaged in any suitable manner.In one embodiment, the pharmaceutical composition is packaged as a kitcontaining a container (such as a vial) of the AdB-2/3 fiber multimer.In a preferred embodiment, the kit further comprises, in the same or aseparate container (such as a vial), a therapeutic, diagnostic, orimaging agent to be administered to a subject, together with the AdB-2/3fiber multimer. Any suitable AdB-2/3 fiber multimer can be used in thekits; in a most preferred embodiment AdB-2/3 fiber multimer is amulitmer (such as a dimer) of JO-1.

In a further aspect, the present invention provides kits comprising (a)one or more recombinant polypeptides/AdB-2/3 fiber multimers, isolatednucleic acids, recombinant expression vectors, and/or host cells of theinvention; and (b) instructions for its/their use in treating a disorderassociated with epithelial tissue. The kits may further comprise atherapeutic for use in the methods of the present invention.

In another aspect, the present invention provides method for identifyingcandidate compounds for one or more of treating a disorder associatedwith epithelial tissue, improving delivery of a substance to anepithelial tissue, for improving delivery of a substance tissueexpressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3infection comprising (a) contacting an AdB-2/3 fiber multimer to DSG2under conditions to promote multimer binding to DSG2, wherein thecontacting is carried out in the presence of one or more test compounds;and (b) identifying positive test compounds that compete with theAdB-2/3 fiber multimer for binding to DSG2 compared to control; whereinthe positive test compounds are candidate compounds for one or more oftreating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, for improving deliveryof a substance tissue expressing DSG2, inducing an EMT in a tissue,and/or treating an AdB-2/3 infection.

Positive test compounds that compete with the AdB-2/3 fiber multimer forbinding to DSG2 are candidate compounds for transiently openingintracellular junctions through their interaction with DSG2. Follow-upassays to verify the ability of the compounds to transiently openintracellular junctions through their interaction with DSG2 can becarried out by any suitable methods, including but not limited tostudies disclosed in the examples that follow. Compounds so identifiedfor treating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, improving delivery of asubstance tissue expressing DSG2, or inducing an EMT in a tissue, can beused as substitutes for the AdB-2/3 multimer in any of the methods ofthe present invention. Furthermore, AdB-2/3 represent important humanpathogens causing respiratory tract infections (some sever) andpharyngoconjunctival fever. Thus compounds that can treat AdB-2/3infection would be useful. As disclosed herein, DSG2 as the primaryhigh-affinity receptor used by AdB-2/3, and thus compounds that candiminish AdB-2/3 binding to DSG2 are candidate compounds for treating orlimiting development of AdB-2/3 infection.

In this aspect, the AdB-2/3 multimer can be any AdB-2/3 multimerdisclosed in any embodiment or combination of embodiments. Thus, invarious non-limiting embodiments, the AdB-2/3 multimer can be AdB-2/3virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd)(subviral dodecahedral particles produced by AdB-2/3 during theirreplication), recombinant AdB-2/3 fiber multimers (including but notlimited to those disclosed in any embodiment or combination ofembodiments below), and functional equivalents thereof. In a preferredembodiment, the one or more AdB-2/3 fiber multimers comprise or consistof an AdB-2/3 PtDd, such as an Ad3 PtDd. In another preferredembodiment, the one or more AdB-2/3 fiber multimers comprise or consistof any embodiment or combination of embodiments of the compositions ofthe invention described below. In a further preferred embodiment, theAdB-2/3 fiber multimer comprises or consists of the a JO-1 (SEQ IDNO:20) dimer, or functional equivalents thereof.

Any suitable control can be used, including but not limited to anAdB-2/3 multimer binding to DSG2 in the absence of test compounds,

In one embodiment, the DSG comprises recombinant DSG2. In anotherembodiment, the methods use cells expressing DSG2 (endogenously orrecombinantly) on the cell surface.

In one non-limiting embodiment, surface plasmon resonance (SPR) studiesusing sensors containing immobilized recombinant DSG2 can be used toidentify candidate compounds that AdB-2/3 fiber multimer binding toDSG2, combined with DSG2 competition studies. Further exemplary studies,such as loss and gain of function analyses, are described in detail inExample 1.

In another embodiment, the identifying comprises transduction studies,where the ability of test compounds to diminish binding is detected as adecrease in the ability of functional AdB-2/3 virions to transduceDSG2-expressing epithelial cells, such as disclosed in Example 1.

In another embodiment, DSG2-expressing cell extract iselectrophoretically separated and Western blotted, and labeled AdB-2/3fiber multimer is used to probe the Western blot in the presence of thetest compounds. In a further embodiment, dot-blot assays can be used,such as those described in Wang et al., J. Virology (2007)81:12785-12792; and Wang et al. (2008) 82:10567-10579.

Further examples of techniques for identifying candidate compounds fortreating an AdB-2/3 infection are provided in the examples that follow.

When the test compounds comprise polypeptide sequences, suchpolypeptides may be chemically synthesized or recombinantly expressed.Recombinant expression can be accomplished using standard methods in theart, as disclosed above. Such expression vectors can comprise bacterialor viral expression vectors, and such host cells can be prokaryotic oreukaryotic. Synthetic polypeptides, prepared using the well-knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Na-amino protected Na-t-butyloxycarbonyl) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Na-amino protected 9-fluorenylmethoxycarbonyl(Fmoc) amino acids. Both Fmoc and Boc Na-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Na-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can bepolyclonal or monoclonal. The antibodies can be humanized, fully human,or murine forms of the antibodies. Such antibodies can be made bywell-known methods, such as described in Harlow and Lane, Antibodies; ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988).

When the test compounds comprise nucleic acid sequences, such nucleicacids may be chemically synthesized or recombinantly expressed as well.Recombinant expression techniques are well known to those in the art(See, for example, Sambrook, et al., 1989, supra). The nucleic acids maybe DNA or RNA, and may be single stranded or double. Similarly, suchnucleic acids can be chemically or enzymatically synthesized by manualor automated reactions, using standard techniques in the art. Ifsynthesized chemically or by in vitro enzymatic synthesis, the nucleicacid may be purified prior to introduction into the cell. For example,the nucleic acids can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the nucleic acids may be used withno or a minimum of purification to avoid losses due to sampleprocessing.

When the test compounds comprise compounds other than polypeptides,antibodies, or nucleic acids, such compounds can be made by any of thevariety of methods in the art for conducting organic chemical synthesis.

Example 1 Desmoglein 2 is a Receptor for Adenovirus Serotypes 3, 7, 11,and 14 Abstract

We have identified desmoglein 2 (DSG2) as the primary high-affinityreceptor used by adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14.These serotypes represent important human pathogens causing respiratorytract infections. In epithelial cells, adenovirus binding to DSG2triggers events reminiscent of epithelial-to-mesenchymal transition,leading to transient opening of intercellular junctions. This improvesaccess to receptors, e.g. CD46 and Her2/neu, which are trapped inintercellular junctions. In addition to complete virions, dodecahedralparticles (PtDd), formed by viral penton and fiber in excess duringviral replication, can trigger DSG2-mediated opening of intercellularjunctions as shown by studies with recombinant Ad3 PtDd. Our findingsshed light on adenovirus biology and pathogenesis and have implicationsfor cancer therapy.

Introduction

Human adenoviruses (Ads) have been classified into six species (A to F)currently containing 55 serotypes. Most Ad serotypes utilize thecoxsackie-adenovirus-receptor (CAR) as a primary attachment receptor¹.This is, however, not the case for species B Ad serotypes. Recently, wehave suggested a new grouping of species B Ads based on their receptorusage². Group 1 (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as areceptor; Group 2 (Ad3, Ad7, 14) share a common, unidentifiedreceptor/s, which is not CD46 and which was tentatively named receptorX; Group 3 (Ad11) preferentially interacts with CD46, but also utilizesreceptor X if CD46 is blocked.

Species B Ads are common human pathogens. Since 2005, a simultaneousemergence of diverse species B serotypes, including serotypes Ad3, Ad7,and Ad14, was observed. In 2007, a new, highly pathogenic and possiblymore virulent strain of Ad14, Ad14a, has been discovered at severalsites in the US and in Asia³⁻⁴. We recently demonstrated that Ad14abelongs to species B group 2 Ads with regards to their receptor usage⁵.Collectively, we will refer to all receptor X-utilizing serotypes (Ad3,Ad7, Ad14, Ad14a, and Ad11) as AdB-2/3.

AdB-2/3 have potential as gene transfer vectors, particularly withregard to tumors of epithelial origin⁶. Epithelial cells maintainseveral intercellular junctions (tight junctions, adherens junctions,gap junctions, and desmosomes), a feature which is often conserved inepithelial cancers in situ and in cancer cell lines⁷. Both CAR and CD46are trapped in intercellular junctions of epithelial cancer cells andare not accessible to Ads that use these attachment receptors⁸⁻⁹. Incontrast, AdB-2/3 efficiently infect epithelial cancer cells, which isaccomplished in part through induction of processes that are reminiscentof Epithelial-to-Mesenchymal Transition (EMT)⁸, a cellulartransdifferentiation program in which epithelial cells losecharacteristics, such as intercellular junctions, and gain properties ofmesenchymal cells¹⁰. Another distinctive feature of AdB-2/3 is theirability to produce subviral dodecahedral particles during theirreplication, consisting of Ad fiber and penton base¹¹.Penton-Dodecahedra (PtDd) cannot assemble from full-length penton baseprotein, but require spontaneous N-terminal truncation by proteolysisbetween residues 37 and 38¹². This cleaved site is conserved in Ad3,Ad7, Ad11, and Ad14 but is not present in Ad2 and Ad5 (FIG. 1 a). In thecase of Ad3, PtDd are formed at a massive excess (5.5×10⁶ PtDd perinfectious virus) and it has been hypothesized that PtDd contribute tovirus escape and spreading¹³.

The first attempts to identify receptor X date back to 1995. Theseinitial studies indicated the interaction of Ad3 with a ˜130 kDa HeLacell protein¹⁴. Recently, several candidates for receptor X such asCD46, CD80 and/or CD86 were suggested¹⁵⁻¹⁸. However, we and others havethus far been unable to verify that these proteins can serve as the highaffinity receptor for AdB-2/3^(2,19-23).

In the present study, using Ad3 virions and recombinant Ad3 PtDd as aprobe for receptor X, we identified desmoglein 2 (DSG2) as a highaffinity receptor for AdB-2/3 serotypes. DSG2 is a calcium-bindingtransmembrane glycoprotein belonging to the cadherin protein family. Inepithelial cells, DSG2 is a component of the cell-cell adhesionstructure²⁴. Its cytoplasmic tail interacts with a series of proteinsthat are in direct contact with regulators of cell adhesion andintercellular junctions/cell morphology²⁵. It has been shown that DSG2is overexpressed in a series of epithelial malignancies includinggastric cancer²⁶, squamous cell carcinomas²⁷, melanoma²⁸, metastaticprostate cancer²⁹, and bladder cancer³⁰.

Results

DSG2 is a receptor for AdB-2/3 viruses. Our previous studies showed thatAd3 binds at nanomolar affinity to a high-density cellular receptor².Ad3 binding was sensitive to trypsin and could be blocked by EDTA,implying that binding required divalent cations. First we sought toidentify the Ad3 capsid protein that mediates high-affinity binding tocells, which we would later use to search for the high-affinity receptorX. Notably, high-affinity binding of Ad5 to CAR and Ad35 binding toCD46, respectively, is mediated by the corresponding fiber knob³¹. Ourprevious studies, however, revealed that a single recombinant, timericAd3 knob could not completely block Ad3 virus binding even when veryhigh concentrations were used, indicating that other or additionalcapsid moieties are involved in Ad3 binding³². Consequently, we utilizedrecombinant Ad3 dodecahedra composed of Ad3 penton bases (BsDd) or Ad3penton bases and fibers (PtDd) (FIG. 1 b)³³ to compete for Ad3 binding.We showed that PtDd but not BsDd blocked attachment of Ad3 to cells(FIG. 2 a). PtDd also blocked binding of other AdB-2/3, e.g. Ad14,Ad14a, as well as Ad11, if CD46 is also blocked. PtDd did, however, notinhibit binding of Ad5 and only partially blocked Ad35 binding (FIG. 2a, FIG. 1 c). Preincubation of cells with PtDd resulted in better Ad3binding inhibition than Ad3 knob mixed with BsDd (FIG. 1 d). The abilityof PtDd to compete with Ad3 was also confirmed in transduction studies,where PtDd efficiently blocked an Ad3 vector (Ad3-GFP) but not thetransduction of an Ad35 vector (Ad35-GFP (that uses CD46 as a receptor)(FIG. 2 b). Ad3-GFP (FIG. 1 e) and Ad35-GFP³⁴ are wild-type Ad3- andAd35-based vectors containing a CMV-GFP expression cassette insertedinto the E3 region.

To select an optimal cell line for receptor X identification, wecompared Ad3 virus binding to several human and animal cell lines (FIG.2 c). Ad3 did not bind to rodent cells suggesting that receptor X wasnot expressed or not accessible to Ad3 in these cells. Of the 10 humancell lines initially tested (HeLa, K562, SKOV3, 293, HT29, SKHep1, Saos,Y79, Ramos), Ad3 binding was absent only on Ramos (human Burkitt'lymphoma) cells.

To identify Ad receptor candidates, HeLa cell membrane proteins weresolubilized, separated on polyacrylamide gels, and blotted. Blots werehybridized with viral particles and binding was visualized with virusfiber knob specific antibodies. Specific gel bands were excised andanalyzed by tandem mass-spectroscopy (MS/MS). First, we tested whetherthis assay can detect a known Ad receptor, CD46. When filters wereincubated with CD46-targeting Ad5/35++virions³⁵, a single band was foundthat matched CD46 (FIG. 2 d). Incubation of filters with Ad3 virionsrevealed two bands with molecular weights of 160 kDa and 90 kDa (FIG. 2e). In addition to these two bands, Ad3 PtDd also reacted with HeLaproteins in the range of 130 kDa. Both 160 and 90 kDa bands were absentin Ramos cells, i.e. cells that do not bind Ad3. The ˜130 kDaPtDd-binding band appeared in both HeLa and Ramos cells suggesting thatit is not an Ad3 virus receptor. MS/MS-analysis of the 160 kDa bandidentified 14 peptides matching human desmoglein 2 (DSG2) (FIG. 2 f).IP/Western analyses of HeLa membrane proteins demonstrated that both the160 and 90 kDa bands were recognized by DSG2-specific antibodies (FIG. 2e, right panel). This is in agreement with previous Western blot studiesshowing that the 160 kDa band represents full size DSG2, and that the 90kDa band is a DSG2 variant that lacks the intracellular domain, thetransmembrane domain, and the juxtamembrane extracellular anchordomain³⁶⁻³⁷.

BIAcore surface plasmon resonance (SPR) studies with sensors containingimmobilized recombinant human DSG2 demonstrated that Ad3, but not Ad2 orAd5, virions interact with DSG2 (FIG. 2g). Recombinant PtDd but not BsDdparticles bound to DSG2 (FIG. 2 h). The K_(D) (equilibrium dissociationconstant) of PtDd-DSG2 interaction was 2.5 nM. PtDd binding toimmobilized DSG2 was specific as demonstrated by the fact that solubleDSG2 competed with it (data not shown). SPR analysis of binding kineticsalso showed that Ad3 fiber knob dissociates faster from DSG2, whichsuggests the existence of additional DSG2 binding site(s) within thefiber shaft and/or the requirement of fiber multimerization for highaffinity binding to DSG2 (FIG. 2 i, also see FIG. 1 d).

Loss- and gain-of-function studies were performed on cell lines tovalidate DSG2 as a critical receptor for AdB-2/3 binding/infection.Recombinant DSG2 protein blocked the binding of Ad3 as well as otherAdB2/3 Ads, i.e. Ad7, Ad14, Ad14a, and Ad11 to HeLa cells, but not thebinding of Ad5 and Ad35 (FIG. 3 a). Ad3-GFP infection was efficientlyinhibited by DSG2 protein but not by other structurally related membersof the cadherin superfamily (desmoglein 1-DSG1 and desmocollin 1-DSC1)³⁸(FIG. 3 b). This study also showed that DSG2 protein had no effect ontransduction by the CD46-targeting vector (Ad35-GFP). Significantinhibition of Ad3 attachment was observed with monoclonal antibodies(mAbs) against extracellular domains 3 and 4 (FIG. 3 c) (for a scheme ofDSG2, see FIG. 2 f). Transfection of HeLa cells with a pool ofDSG2-specific siRNAs resulted in ˜7-fold downregulation of surface DSG2levels (data not shown). Ad3 attachment was 3 fold lower in DSG2-siRNAtreated HeLa cells compared to control siRNA-treated cells (p<0.001)(FIG. 3 d). GFP expression levels after infection with Ad3-GFP were13.9-fold lower in DSG2-siRNA transfected cells than in control siRNAtransfected cells (FIG. 3 e). DSG2-specific siRNA did not affecttransduction with the CAR-targeting vector Ad5-GFP. However, DSG2-siRNAtransfection also decreased binding and transduction with theCD46-specific vectors Ad35-GFP and Ad5/35-GFP. DSG2-siRNA did notdecrease CD46 levels in HeLa cells. At this point we cannot explain thisphenomenon. It appears, however, to be specific to HeLa cells. Nodecrease in Ad35-GFP or Ad5/35-GFP transduction was detected in 293cells or breast cancer BT474 cells that were transfected with DSG2-siRNA(data not shown).

siRNA mediated DSG2 downregulation also decreased viral cytolysis andspread in cells that were infected at 100% confluence at an MOI of oneAd3-GFP pfu per cell. Using adjusted multiplicities of infection (MOIs)(to achieve comparable percentages of GFP expression at 16 hourspost-infection), we followed viral cytolysis over time and found largerlytic plaques in control siRNA than in DSG2 siRNA-transfected cells atday 7 p.i. This is reflected in crystal violet staining of viable cellsthat remained attached to tissue culture wells, i.e. cells that did notdevelop cytopathic effects due to virus infection (FIG. 3 f).Quantitative analysis of cell viability showed significantly less cellkilling in cells in which DSG2 was downregulated by siRNA compared tocontrol siRNA treated cells (FIG. 3 g).

For gain-of-function studies, we selected a series of cell lines withdifferent DSG2 expression levels and measured Ad3-GFP transduction (FIG.4 a). All cell lines that lacked DSG2 expression (lymphoma Ramos, Raji,Mino, and HH cells) were refractory to Ad3-GFP transduction, but couldbe transduced by the CD46 targeting Ad5/35-GFP vector (because CD46 isexpressed on these cells³⁹). DSG2-positive K562 cells, on the otherhand, could be efficiently transduced with Ad3-GFP. About 70% of BJABcells were DSG2 positive and, correspondingly, the percentage ofGFP-positive cells reached a plateau at about 50%. To conclusively provethe critical role of DSG2 in Ad3 infection, we ectopically expressedDSG2 via lentivirus vector gene transfer in the histiocytic lymphomacell line U937, which is refractory to Ad3-GFP transduction (FIG. 4 b).Ectopic DSG2 expression in U937-DSG2 cells conferred efficient Ad3attachment and transduction, whereas Ad35 attachment and Ad5/35-GFPtransduction was unaffected in these cells (FIGS. 4 c and d).

DSG2 localization in human cells: As expected, we found DSG2 in cellmembranes of normal epithelial tissues (foreskin and colon) andepithelial cancers (breast and ovarian cancer) (FIG. 5 a). Confocalimmunofluorescence microscopy studies of polarized colon cancer T84 andCaCo-2 cells demonstrated colocalization of DSG2 and the intercellularjunction protein Claudin 7 (FIG. 5 b). In stacked XZ image sections(FIG. 5 b) {or XY sections taken at different depth of the cell layer(data not shown), DSG2 appears at the distal end of intercellularjunctions. DSG2 also colocalized with the adherens junction proteinE-cadherin in epithelial cells ( ). Fifteen minutes after addingCy3-labelled Ad3 to polarized cells, viral particles were detectable inassociation with junction-localized DSG2 (FIG. 5 c). Similar resultswere obtained with normal small airway epithelial cells incubated withPtDd for 15 min as shown by triple labeling of Cy5-PtDd, DSG2, andE-cadherin (FIG. 5 d, upper panel). PtDd signals were on cell membranes(FIG. 5 d, lower panel, thin arrows) and in the cytoplasm (thickarrows), most likely reflecting internalized particles.

In contrast to polarized epithelial cell lines, in non-polarized cells,such as HeLa cells, intercellular junctions (i.e. membrane-localizedClaudin 7 and E-cadherin signals) were absent. DSG2 and Ad3 were founddispersed over the cell surface (FIG. 5 e).

Our studies on Ad3 infection of HeLa cells (FIGS. 2 and 3) indicate thatDSG2 also acts as receptor in non-polarized cells. In this context, itis noteworthy that we detected DSG2 and Ad3 binding/transduction inhuman platelets and granulocytes (data not shown). Although thesefindings are relevant for Ad3 pathogenesis and the intravascularapplication of Ad3 vectors for gene therapy purposes, we focused in thisstudy on analyzing the consequences of Ad3-DSG2 interaction in polarizedepithelial cells.

Ad3 interaction with DSG2 triggers EMT. Recently, we found that AdB-2/3interaction with epithelial ovarian cancer cells triggered EMT. EMT ischaracterized by increased expression of mesenchymal markers, increasedexpression of extracellular matrix compounds, decreased expression ofepithelial markers, altered location of transcription factors, andactivation of kinases⁷. Here, we attempted to prove that Ad3 interactionwith DSG2 triggers EMT-like events. To avoid potential side effects ofviral gene expression on cell morphology, the studies utilizedultraviolet light (UV)-inactivated Ad particles and recombinant Ad3PtDd. Overall, the results with both types of particles were similar.Incubation of epithelial cancer cells with PtDd (FIG. 6) orUV-inactivated Ad3 (not shown) caused remodeling of junctions asreflected by the decrease in of membrane/junction-localized Claudin 7(FIG. 6 a) or E-cadherin signals (FIG. 6 b). Furthermore, after PtDdtreatment we found stronger immunofluorescence signals of themesenchymal markers Vimentin and Lipocalin 2 (FIGS. 6 c and d). Toidentify intracellular signaling pathways triggered by PtDd interactionwith DSG2, we studied mRNA expression profiles. Twelve hours afterincubation of polarized BT474 cells with PBS, BsDd, or PtDd, mRNA wasanalyzed using Affymetrix human ST gene arrays. We found that PtDdtreatment resulted in >1.5-fold upregulation of 430 genes and >1.5-folddown-regulation of 352 genes when compared to PBS-treated cells (FIG. 6e). The list of altered genes was further processed by Pathway-Expresssoftware⁴⁰. This computation suggested that PtDd mediated markedactivation of a number of signaling pathways involved in EMT, includingphosphatidylinositol (PI), mitogen activated protein kinase (MAPK akaERK), Wnt, adherens junctions, focal adhesion, and regulation of actincytoskeleton signaling pathways.

Western blot analysis using phosphorylation-specific antibodies showedthat PtDd, but not BsDd, triggered the activation of PI3K andMAPK/ERK1/2, i.e. key kinases involved in EMT (FIG. 6 f). Activation ofthese pathways was also triggered by DSG2-specific mAbs (6D8, and to alesser degree 10D2, and 13B11,) but not with mAbs directed against CD46.PtDd activation of pathways was mediated by DSG2, because MAPK/ERK1/2and PI3K phosphorylation was decreased in cells transfected with DSG2siRNA, but not in control siRNA treated cells. Finally, PtDd-triggeredphosphorylation of kinases was absent when cells were pretreated withthe ERK1/2 inhibitor UO126 (upper panel) or the PI3K inhibitorWortmannin (lower panel). Taken together, our data suggest that Ad3 orAd3PtDd binding to DSG2 triggers EMT in epithelial cells.

Ad3 and PtDd increase access to receptors that are trapped inintercellular junctions. To test whether Ad3 virion- or Ad3PtDd-triggered EMT also results in opening of intercellular junctions,we studied barrier properties in monolayers of epithelial cells. Firstwe measured the flux of 4 kDa FITC-dextran through confluent polarizedBT474 cells cultured in transwell chambers (FIG. 7 a). We found thatPtDd but not BsDd incubation significantly increased the permeabilitycoefficient compared to phosphate buffered saline (PBS). We then testedwhether Ad3 or PtDd-triggered EMT and transient junction-opening wouldincrease access to proteins that are normally not accessible due toepithelial cell junctions. An example for such a junction-localizedreceptor is CD46, the high-affinity receptor for Ad35 and Ad5/35⁸. Weconfirmed that a large number of CD46 molecules localizes to junctionsof BT474 cells (FIG. 7 b, left panels). PtDd pre-treatment significantlyincreased the attachment of ³H-Ad35 to BT474 cells when compared to BsDdtreatment (FIG. 7 b, right panel). An enhancing effect of PtDd ontransduction of CD46-targeting Ad vectors was also demonstrated in vivoin subcutaneous epithelial tumors (FIG. 7 c). Intravenous injection ofPtDd eight hours before application of Ad5/35-bGal increased viraltransduction. Beta-galactosidase activity, measured in tumor lysates 3days after Ad injection, was 2.3(+/−0.2)×10⁵ rlu/μg protein,2.7(+/−0.6)×10⁵ rlu/μg, and 38(+/−3.5)×10⁵ rlu/μg for mice that weremock-injected, BsDd-coinjected, and PtDd-coinjected, respectively.

In another line of experiments in breast cancer cell cultures, we foundthat Her2/neu, the receptor for the widely used monoclonal antibodyHerceptin (trastuzumab) co-stained with the intercellular junctionprotein Claudin 7 (FIG. 7 d). This suggests that not all Her2/neumolecules are accessible to Herceptin. Incubation of theHer2/neu-positive breast cancer cell line BT474 with PtDd triggeredrelocalization of Her2/neu to the cell surface (FIG. 7 e). Toconsolidate this observation, we tested whether Ad3 or Ad3PtDd wouldimprove BT474 cell killing by Herceptin. In agreement with earlierstudies⁴¹, Herceptin caused death of approximately 25% of BT474 cellscells (FIG. 7 f). Pre-incubation of BT474 cells with UV-inactivated Ad3particles or PtDd increased Herceptin cytotoxicity by more than 2-fold.Incubation with UV-inactivated Ad5 particles or BsDd had no effect onHerceptin killing. In addition, Herceptin and PtDd/Herceptin had nocytotoxic effect on the Her2/neu-negative breast cancer cell lineMDA-MB-231 (FIG. 8 a). The enhancing effect of PtDd and Ad3 on Herceptinkilling of BT474 cells was mediated by DSG2, as downregulation of DSG2in BT474 cells by DSG2 siRNA abolished this effect (FIG. 7 g). We alsostudied whether inhibition of key pathways involved in EMT affects theenhancing effect of PtDd on Herceptin cytotoxicity. These studies showedthat inhibition of PI3K by Wortmannin, as well as inhibition of MAPK/ERKby U0126 counteracted PtDd enhancement of Herceptin therapy (FIG. 7 g).Importantly, intravenous injection of PtDd (2 mg/kg) intoBT474-M1-tumor-bearing mice before Herceptin treatment resulted inelimination of tumors, an outcome that could not be achieved withHerceptin injection alone (FIG. 7 h). PtDd pretreatment also allowed theunfolding of the therapeutic effect of the EGFR-specific mAb Erbitux(cetuximab) as it increased killing of EGFR-positive colon cancer LoVocells with this antibody in vitro (FIG. 8 b).

Discussion

In this study we describe two major findings: i) DSG2 is a receptorcrucial for infection of a series of human adenoviruses that are commonpathogens and important biomedical tools. ii) Ad interaction with DSG2results in the opening of intercellular junctions, thus increasingaccess to receptors trapped therein.

DSG2 is an Ad attachment receptor. The use of complete Ad3 particles orPtDd as a receptor probe was instrumental in the identification of DSG2as the attachment receptor for Ad3, Ad7, Ad11, and Ad14. Previousattempts with Ad3 fiber knob domains as a bait did not yield meaningfulreceptor candidates³². Our competition and surface plasmon resonancestudies shown in FIG. 2, indicate that the DSG2 interacting domain(s)within Ad3 are formed by the fiber only in the spatial constellationthat is present in viral particles. This clearly widens ourunderstanding of Ad attachment mechanisms, which, so far, were thoughtto involve only a high affinity interaction between the fiber knob andthe cellular receptor, i.e. CAR or CD46.

Role of Ad3 and PtDd interaction with DSG2 in viral dissemination.During replication of Ad5, excess production of fiber results in thedisruption of epithelial junctions either by interfering with CARdimerization (which is critical for maintenance of junctions) or bytriggering intracellular signaling that leads to reorganization ofintercellular junctions⁴²⁻⁴³. Both mechanisms could also be involved inAd3 virion/DSG2- and Ad3 PtDd/DSG2-mediated intercellular junctionopening. We have experimental support for intracellular signalingtriggered by Ad3 and PtDd binding to DSG2 in epithelial cells.Immunofluorescence, PI3K/MAPK phosphorylation, mRNA expression array,and metabolic pathway inhibition data suggest that Ad3 and PtDd triggerEMT in epithelial cells resulting in transient opening of intercellularjunctions. Intercellular junction opening mediated by interaction of Ad3particles or recombinant PtDd with DSG2 is further supported byincreased cell permeability and access to receptors that are trapped inintercellular junctions (CD46 and Her2/neu). Along this line, a recentstudy showed that antibodies against the extracellular domain of DSG2resulted in the opening of intercellular junctions in CaCo-2 cellmonolayers⁴⁴.

Ad3 virion- and PtDd-triggered EMT, i.e. the dissociation of theintercellular junctions, appears to have an important biological role.We speculate that, specifically the massive overproduction of PtDdduring viral infection and its interaction with DSG2, facilitate thelateral viral spread in epithelial cells and, potentially, thepenetration of Ad into subepithelial cell layers and the blood stream.

Implications for Ad pathogenesis. Our findings that DSG2 is anattachment receptor that facilitates further viral spread, sheds lighton the Ad3 infection mechanism of the respiratory tract epithelium.Furthermore, the observation that Ad3 binds to DSG2 on platelet andgranulocytes has potential implications on systemic spread of this virusonce it has entered the blood stream. Although mouse DSG2 shares 76%homology with human DSG2⁴⁵, our data, showing that mouse cells arerefractory to Ad3-GFP infection, indicate that mouse DSG2 cannotfunction as an Ad3 receptor. To study pathogenesis of AdB-2/3 serotypes,transgenic mice that express human DSG2 under the control of adequateendogenous regulatory elements resulting in DSG2 expression in a patternand at levels similar to humans, would be a critical tool.

Implications for cancer therapy. DSG2 has been proposed as a marker forepithelial tumors⁴⁶. The epithelial phenotype of cancer cells and theability to form physical barriers represent a mechanism that restrictsaccess of drugs, antibodies, oncolytic viruses, or immune cells to thesites of tumors, thus diminishing the efficacy of such therapeuticmodalities⁴⁷. We demonstrated here, in three examples (Ad5/35 vectors,Herceptin, and Erbitux), that this important problem in cancer therapycan perhaps be addressed by the use of DSG2-interacting Ad3 components.

In conclusion, we report the discovery of the high affinity receptor fora series of common human Ads. Our study contributes to the understandingof how Ads induce cellular processes in order to gain access toepithelial tissue. Our findings have implications for improving cancertherapies.

Methods

Proteins and antibodies. The knob domains of Ad3, Ad5, and Ad35 fiberswere produced in E. coli as described elsewhere⁴⁸. Recombinant Ad3penton-dodecahedra (PtDd) and base dodecahedra (BsDd) were produced ininsect cells and purified as described previously³³. Polyclonal rabbitantibodies against purified recombinant Ad3 and Ad35K++knob wereproduced by PickCell Laboratories B. V. (Amsterdam, The Netherlands).DSG2-specific monoclonal antibodies 20G1, 7H9, 13B11, 10D2 and 8E5⁴⁹were purified from hybridoma culture supernatant.

Cell lines. Cells were cultured as described in the SI. BT474 is a Hepositive breast cancer cell line with epithelial cell features. Toachieve cell polarization, 1.4×10⁵ BT474, T84 and CaCo-2 cells werecultured in collagen-coated 6.5 mm Transwell inserts (0.4 μm pore size)(Costar Transwell Clears) for 10 days until transepithelial resistancewas stable.

Adenoviruses. Wild-typeAd3 (GB strain), Ad7p (Gomen stain), Ad11p(Slobitski strain), Ad14 (DeWit strain), and Ad35 (Holden strain) wereobtained from the ATCC. Ad14a is new genomic variant of Ad14⁵.Propagation, methyl-³H thymidine labeling, purification and titering ofAds was performed as described elsewhere². Ad5/35-GFP and Ad5-GFP areAd5 vectors containing Ad35 and Ad5 fibers and a CMV-GFP expressioncassette⁵⁰. Ad3-GFP and Ad35-GFP are wild-type Ad3- and Ad35-basedvectors containing a CMV-GFP expression cassette inserted into the E3region. Construction of Ad3-GFP is described in SI. Ad35-GFP has beendescribed previously³⁴. For transduction studies, cells were exposed toAd vectors at the indicated MOIs for one hour, washed, and GFPexpression was measured by flow cytometry 18 hours later.

Membrane protein preparation. HeLa cell membrane proteins were preparedas described earlier⁵¹. Briefly, HeLa cell pellets were re-suspended inice-cold homogenization buffer (20 mM Hepes, 1.5 mM MgCl₂, 5 mM KCl, 150mM NaCl, 15% glycerol, 0.25 M sucrose, 0.1 mM EDTA, 2 mMβ-mercaptoethanol, 1 mM PMSF). After disruption with a 3 ml syringe and21G needle, the lysate was centrifuged at 400×g for 15 minutes. Thesupernatant was diluted with 2 times volume of PBS and centrifuged at35,000 rpm for 1 hour in a Beckman ultracentrifuge. The membrane proteinpellet was resuspended in solubilization buffer (50 mM Hepes, 5 mMMgCl₂, 5 mM KCl, 150 mM NaCl, 15% glycerol, 0.25 M sucrose, 0.1 mM EDTA,2 mM β-mercaptoethanol, 1 mM PMSF, 0.5% Brij 96V (Fluka, St Louis, Mo.).The use of Brij96V as a detergent was instrumental as desmosomalproteins are highly insoluble.

Western blot with Ad3 and PtDd. Technical details for mass-spectroscopyanalysis are described elsewhere⁵¹. To immunoprecipitate DSG2 fromsoluble crude membrane protein preparations, DSG2-specific mAb 6D8, andthe Pierce Crosslink Immunoprecipitation Kit (Pierce Biotechnology,Rockford, Ill.) were used. Crude membrane proteins from HeLa cells weresolubilized with 0.5% detergent Brij 96V, pre-incubated with controlresin for 3 hours at 4° C. to reduce nonspecific binding, and thenincubated with DSG2 antibody crosslinked proteinA/G agarose overnight at4° C. Bound protein were eluted per manufacturer's instruction.

Surface plasmon resonance (SPR) analyses. Acquisitions were done on aBIAcore 3000 instrument. HBS-N (GE-Healthcare, Pittsburgh, Pa.)supplemented with 2 mM CaCl₂ was used as running buffer in allexperiments at a flow rate of 5 μl min⁻¹. Immobilisation on CM4sensorchip (BIAcore) was performed using DSG2 (Leinco Technology, Inc.)at 0.1 mg ml⁻¹ diluted in 10 mM sodium acetate buffer pH4.2 injected for10 minutes on EDC-NHS activated flow-cell. A control flow-cell wasactivated by EDC-NHS and inactivated by ethanolamine. Differentconcentration of PtDd, BsDd, Ad3 fiber knob were injected for 5 minutesfollowed by 3 minutes dissociation time and the signal was automaticallysubtracted from the background of the ethanolamine deactivated EDC-NHSflow cell. For the adenovirus binding experiments, a similar protocolwas used with the injection of wild-type Ad2, Ad3 and Ad5 at 5·10⁹ vpper ml.

siRNA studies. A set of DSG2 specific siRNA was synthesized by Dharmacon(Thermo Scientific). The target sequences were CAAUAUACCUGUAGUAGAA (SEQID NO: 29), GAGAGGAUCUGUCCAAGAA (SEQ ID NO: 30), CCUUAGAGCUACGCAUUAA(SEQ ID NO: 31) and CCAGUGUUCUACCUAAAUA (SEQ ID NO: 32). Control siRNAwas purchased from Qiagen, Valencia, Calif. siRNA transfection wasperformed using HyperFect transfection reagent (Qiagen).

DSG2 expressing 0937 cells. DSG2 cDNA (accession No. BC099657) fromCapital Biosciences (Rockville, Md.) was cloned into the lentivirusvector pRRL-SIN⁵² under the control of the EF1α, promoter.VSVG-pseudotyped lentivirus vectors were produced and titered asdescribed earlier⁵³.

Animal studies: All experiments involving animals were conducted inaccordance with the institutional guidelines set forth by the Universityof Washington. Mice were housed in specific-pathogen-free facilities.Breast cancer xenografts were established by injecting cancer cells inmatrigel (1:1 vol/vol) into the mammary fat pad of CB17 SCID-beige mice.Herceptin was injected intraperitoneally at a dose of 10 mg/kg. PtDd wasgiven intravenously at a dose of 2 mg/kg. Tumor volumes were measured asdescribed previously⁵⁴.

References for Example 1

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Example 2 Multimerization of Adenovirus Serotype 3 Fiber Knob Domains isRequired for Efficient Binding of Virus to Desmoglein 2 and SubsequentOpening of Epithelial Junctions Abstract

In Example 1, we identified desmoglein 2 (DSG2) as the main receptor fora group of species B adenoviruses (Ads), including Ad3, a serotype thatis widely distributed in the human population. In this example, we haveattempted to delineate structural details of Ad3 interaction with DSG2.For CAR- and CD46-interacting Ad serotypes, attachment to cells can becompletely blocked by an excess of recombinant fiber knob protein, whilesoluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We foundthat the DSG2 interacting domain(s) within Ad3 are formed by severalfiber knob domains that have to be in the spatial constellation that ispresent in viral particles. Based on this finding, we generated a smallrecombinant, self-dimerizing protein containing the Ad3 fiber knob(Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blockedAd3 infection. We demonstrated by confocal immunofluorescence andtransmission electron microscopy analyses that Ad3-K/S/Kn, through itsbinding to DSG2, triggered transient opening of intercellular junctionsin epithelial cells. Pretreatment of epithelial cells with Ad3-K/S/Knresulted in increased access to receptors that are localized in ormasked by epithelial junctions, e.g. CAR or Her2/neu. Ad3-K/S/Kntreatment released CAR from tight junctions and thus increasedtransduction of epithelial cells by a serotype Ad5-based vector.Furthermore, pretreatment of Her2/neu-positive breast cancer cells withAd3-K/S/Kn increased killing of cancer cells by the Her2/neu-targetingmonoclonal antibody trastuzumab (Herceptin). This study widens ourunderstanding of how Ads achieve high avidity to their receptors andinfection of epithelial tissue. The small recombinant protein Ad3-K/S/Knhas practical implications for the therapy of epithelial cancer andgene/drug delivery to normal epithelial tissues.

Introduction

The protruding fiber is the moiety within the Ad capsid that mediates ahigh affinity binding to the primary attachment receptor. Each Ad capsidhas 12 fibers linked to penton bases. Each fiber consists of a taildomain that is anchored within the penton base, a shaft domainconsisting of repeats of up to 14 amino acids that form β-sheets (withthe number of repeats ranging from 6 to 23 in different serotypes), andthe C-terminal homo-trimeric knob domain. For CAR- and CD46-interactingAds, the knob domain binds with high affinity to the receptor andsoluble fiber knobs completely block infection.

In this study, we have attempted to delineate structural details of Ad3interaction with DSG2. We report that multimers of (trimeric) Ad3 fiberknobs are required for high affinity DSG2 binding. This is clearlydifferent from the strategy of CAR- and CD46-interacting Ad serotypes toachieve infection. This specific mode of Ad3-DSG2 interaction isfunctionally crucial for Ad3, because it allows opening of epithelialjunctions.

Material and Methods

Proteins and antibodies. Recombinant human DSG2 protein was from LeincoTechnologies, Inc. (St. Louis, Mo.). The recombinant Ad3 fibers wereproduced in E. coli with N-terminal 6-His tags, using the pQE30expression vector (Qiagen, Valencia, Calif.) and purified by Ni-NTAagarose chromatography as described elsewhere (34). Recombinant Ad3penton-dodecahedra (PtDds) was produced in insect cells and purified asdescribed previously (8).

The following antibodies were used for immunofluorescence studies:polyclonal goat anti-D562 (R&D Systems, Inc., Minneapolis, Minn.), mousemAb anti-DSG2 (clone 6D8) (Cell Sciences, Canton, Mass.), rabbitanti-Claudin 7 (abcam, Cambridge, Mass.), FITC conjugated goatanti-adenovirus (Millipore Billerica, Mass.), monoclonal anti-6×His(Serotec, MCA1396), rabbit anti-ZO-1 antibody (Cell Signaling TechnologyInc., Beverly, Mass.). Polyclonal rabbit antibodies against purifiedrecombinant Ad3 knob were produced by PickCell Laboratories B. V.(Amsterdam, The Netherlands). Monoclonal anti-DSG2 antibodies 20G1, 7H9,13B11, 10D2 and 8E5 (12) were purified from hybridoma culturesupernatants.

Recombinant Ad3 fiber knobs. The recombinant Ad3 fiber knobs S/Kn,S2/Kn, S3/Kn, S4/Kn, S5/Kn, and S6/Kn were generated by PCR using Ad3genomic DNA as a template. The PCR products were then cloned into the E.coli expression vector pQE30 as BclI/HindIII or BamHI/HindIII fragments.The following primers were used:

S6/Kn-forward: (SEQ ID NO: 33)5′-CTGATGAATTCTTGATCAGGGGTTTTAAGTCTTAAATGTGTTAAT CC-3′ S5/Kn-forward:(SEQ ID NO: 34) 5′-TTACTGATGAATTCTTGATCA GGCTCCCTCCAACTTAAAGTGGGAAGTGGT-3′ S4/Kn-forward: (SEQ ID NO: 35)5′-TTACTGATGAATTCTGGATCC TTAGAAGAAAACATCAAAGTTAA CAC-3′ S3/Kn-forward:(SEQ ID NO: 36) 5′-TTACTGATGAATTCTGGATCC CATTCTATAAATTTACCAATAGGAAACGGT-3′ S2/Kn-forward: (SEQ ID NO: 37)5′-TTACTGATGAATTCTGGATCC AACAAACTTTGCAGTAAACTCGGA AATGG-3′ S/Kn-forward:(SEQ ID NO: 38) 5′-ACCATCACGGATCCAATTCTATTGCACTGAA-3′reverse for all constructs: (SEQ ID NO: 39)5′-AGCTAATTAAGCTTAGTCATCTTCTCTAATATAGG-3′

For generating the K- or E-coil containing Ad3 fiber knobs the followingoligonucleotides were annealed and cloned into pQE30 as BamHI fragments.

for pQE30-Kcoil:

(SEQ ID NO: 40) 5′ATCAAAGGTAAGCGCTTTAAAGGAGAAAGTTTCAGCACTTAAAGAAAAGGTATCCGCTTTAAAGGAGAAAGTTTCAGCACTTAAAGAAAAAGTG TCCGCTCTGAAAGAAG-3′ and(SEQ ID NO: 41) 5′GATCCTTCTTTCAGAGCGGACACTTTTTCTTTAAGTGCTGAAACTTTCTCCTTTAAAGCGGATACCTTTTCTTTAAGTGCTGAAACTTTCTC CTTTAAAGCGCTTACCTTT-3′for pQE30-Ecoil (SEQ ID NO: 42)5′ATCAGAGGTAAGCGCTTTAGAGAAAGAAGTTTCAGCACTTGAGAAGGAGGTATCCGCTTTAGAGAAAGAAGTTTCAGCACTTGAGAAGG AAGTGTCCGCTCTGGAAAAAG-3′and (SEQ ID NO: 43) 5′GATCCTTTTTCCAGAGCGGACACTTCCTTCTCAAGTGCTGAAACTTCTTTCTCTAAAGCGGATACCTCCTTCTCAAGTGCTGAAACTTCT TTCTCTAAAGCGCTTACCTCT-3′

To generate Ad3-E/S2/Kn and Ad3-K/S2/Kn, the following primers wereused:

Forward: (SEQ ID NO: 44)5′ATCTAGGATCCGGTGGCGGTTCTGGCGGTGGCTCCGGTGGCGGTTCTAACAAACTTTGCAGTAAACTCGGAAATGGTCTTACATTTGACT-3′ Reverse: (SEQ ID NO: 45)5′AGCTAATTAAGCTTAGTCATCTTCTCTAATATAGG-3′

The PCR products were then cloned into the BamHI/HindIII site ofpQE30-Kcoil and pQE30-Ecoil.

To generate Ad3-E/S/Kn and Ad3-K/S/Kn, the following primers were used:

Forward: (SEQ ID NO: 46)5′TTATTGCTACTGGATCCGGTGGCGGTTCTGGCGGTGGCTCCGGTGGCGGTTCTAATTCTATTGCACTGAAAAATAACAC-3′ Reverse: (SEQ ID NO: 47)5′AGCTAATTAAGCTTAGTCATCTTCTCTAATATAGG-3′

The PCR products were than cloned into the BamHI/HindIII site ofpQE30-Kcoil and pQE30-Ecoil.

Cell lines. 293 (Microbix, Toronto, Ontario, Canada), HeLa (AmericanType Culture Collection, ATTC) were cultured in Dulbecco modified Eaglemedium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol/LL-glutamine (Glu), 100 units/mL penicillin, and 100 μg/mL streptomycin(P/S). BT474-M1 cells (16) were cultured in DMEM/F:12 with 10% FBS, 1%Pen/Strep and L-Glutamine. Colon cancer T84 cells (ATCC CCL-248)) werecultured in a 1:1 mixture of Ham's F12 medium and DMEM, 10% FBS, Glu andP/S. To achieve cell polarization, 1.4×10⁵ T84 cells were cultured in6.5 mm Transwell inserts (0.4 μm pore size) (Costar Transwell Clears)for more than 20 days until transepithelial resistance was stable.

Adenoviruses. Propagation, methyl-³H thymidine labeling, purificationand titering of Ads was performed as described elsewhere (31).Ad5/35-GFP and Ad5-GFP are Ad5 vectors containing Ad35 and Ad5 fibersand a CMV-GFP expression cassette (24). Ad3-GFP is a wild-type Ad3-basedvector containing a CMV-GFP expression cassette inserted into the E3region (33). Ad5/3L-GFP and Ad5/3S-GFP are EVE3 deleted, Ad5-basedvectors, containing the same CMV-GFP expression cassette inserted intothe E3 region. The construction of chimeric Ad vectors followed theprotocol described earlier (23). The primer sequences used to constructthe chimeric fiber genes were as follows: for Ad5/3L-GFP, SF(5′-GACACGGAAACCGGTCCTCCAACTGTGCCTTTTCTTACTCC-3′) (SEQ ID NO: 48), SR(5′-GCAGTTGGCTTCTGGTTTTGGACCTGTCCACAAAGTTAGCTTATCATTATTTTTGTTTCC-3′)(SEQ ID NO: 49), KF(5′-GGAAACAAAAATAATGATAAGCTAACTTTGTGGACAGGTCCAAAACCAGAAGCCAACTGC-3′)(SEQ ID NO: 50), KR(5′-TGAAAAATAAACACGTTGAAACATAACACAACTAGTTCTTTATTCTTGGGCATTTTAGTCATCTTCTCTAATATAGGAAAAGGTAAATG-3′) (SEQ ID NO: 51), R1(5′-CATTTACCTTTTCCTATATTAGAGAAGATGACTAAAATGCCCAAGAATAAAGAACTAGTTGTGTTATGTTTCAACGTGTTTATTTTTCA-3′) (SEQ ID NO: 52) and R2(5′-ATACTTAGGGTACCAATCGATATGGCCACGTGGGTTCTGTGGTCCC-3′) (SEQ ID NO: 53).For Ad5/3S-GFP, SF(5′-ACGGAAACCGGTCCTCCAACTGTGCCTTTTCTTACTCCTCCCTTTGTATCCCCCAATGGGTTTCAAGAGAGTCCCCCTGGGGTTTTAAGTCTTAAATGTG-3′) (SEQ ID NO: 54), KR(5′-GAAAAATAAACACGTTGAAACATAACACACTCGAGTCTTTATTCTTGGGCATTTTAGTCATCTTCTCTAATATAGGAAAAGGTAAATG-3′) (SEQ ID NO: 55), R1(5′-CATTTACCTTTTCCTATATTAGAGAAGATGACTAAAATGCCCAAGAATAAAGACTCGAGTGTGTTATGTTTCAACGTGTTTATTTTTC-3′) (SEQ ID NO: 56) and R2(5′-ATACTTAGGGTACCAATCGATATGGCCACGTGGGTTCTGTGGTCCC-3′) (SEQ ID NO: 57).SpeI or XhoI restriction sites were introduced after the fiber stopcodon for Ad5/3L-GFP or Ad5/3S-GFP, respectively. To generatefull-length E1/E3-deleted vector genomes, the corresponding shuttleplasmid containing chimeric fiber genes and GFP expression cassette wereinserted in pAdHM4 by homologous recombination in E. coli strain BJ1583.The resulting plasmids pAd5/3L-GFP and pAd5/3S-GFP were analyzed byrestriction analysis and sequencing. To produce the correspondingviruses, pAd5/3L-GFP and pAd5/3S-GFP were digested with PacI to releasethe viral genomes and transfected into 293 cells as described before.Recombinant viruses were propagated in 293 cells and purified bystandard methods. Ad particle (viral particle, VP) concentrations weredetermined spectrophotometrically by measuring the optical density at260 nm (OD₂₆₀) and plaque titering (plaque forming units, pfu) wasperformed using 293 cells as described elsewhere (24). The VP to pfuratio was 20:1 for all virus preparations.

Attachment and transduction assays. Adherent cells were detached fromculture dishes by incubation with Versene and washed with PBS. A totalof 1.8×10⁵ cells/tube was resuspended in 100 μl of ice-cold adhesionbuffer containing ³H-labeled Ad at a multiplicity of infection (MOI) of8,000 VP per cell. After 1 h of incubation at 4° C., cells were pelletedand washed twice with 0.5 ml of ice-cold wash-buffer (PBS, 1% FBS).After the last wash, the supernatant was removed, and thecell-associated radioactivity was determined with a scintillationcounter. The number of viral particles (VP) bound per cell wascalculated by using the virion specific radioactivity and the number ofcells. For competition studies, 4.5 μg of competitor (PtDds, fiberknobs, antibodies) were allowed to attach for 60 min at 4° C. inadhesion buffer and non-bound competitor removed by washing cells twicewith PBS before cells were resuspended in attachment buffer containing³H-labeled Ad. For transduction studies, cells were exposed to Advectors at the indicated MOIs for one hour, washed, and GFP expressionin 20,000 cells was measured by flow cytometry 18 hours later.

Ad5 infection studies. In all transduction studies of HeLa, we have usedconditions (MOI, virus concentration, exposure time) that have beenoptimized previously to be within the linear range of transduction (33).

Western blot: Mini-PROTEAN precast gels (BIO-RAD, Hercules, Calif.) with4-15% gradient polyacrylamide were used. For the studies shown in FIG.2B, a total of 0.5 μg protein was mixed with 2× loading buffer (10 mMTris-HCl, pH6.8, 200 mM DTT, 4% SDS, 20% glycerol, 0.2% bromophenolblue). Samples were either boiled (B) for 5 min or loaded unboiled (UB).The following running buffer was used: 25 mM Tris, 0.192 M glycine, 0.1%SDS, pH8.3. After electrophoresis, proteins were transferred tonitrocellulose and incubated with anti-DSG2 antibodies or anti-Ad3knobantibodies as described previously (33).

Native polyacrylamide gel electrophoresis: For the study shown in FIG.2F, a total of 0.4 μg proteins were mixed in 2× sample buffer (62.5 mMTris-HCl, pH6.8, 40% glycerol, 0.01% bromophenol blue), not boiled, andrun in 25 mM Tris, pH 8.3, 0.192 M glycine.

Permeability assay. A total of 5×10⁵ T84 cells were seeded in 12 mmtranswell inserts (PET membrane, with 0.4 μm pore size (Corning, N. Y.)and cultured for >20 days until transepithelial resistance was stable.Culture medium was changed every 2-3 days. The cells were exposed toDSG2 ligands (20 μg/ml) in adhesion medium (DMEM, 1% FBS, 2 mM MgCl₂, 20mM HEPES) for 15 min at room temperature. Then, 1 mCi of [¹⁴C]polyethylene glycol-4000 (PEG-4000); (Perkin Elmer, Covina Calif.)diluted with DMEM/K12 medium was added into the inner chamber. Mediumaliquots were harvested from the inner and outer chamber at 15 and 30min and measured by a scintillation counter. Permeability was calculatedas described elsewhere (36).

Trastuzumab cytotoxicity assay. 5×10⁴ BT474-M1 cells/well were plated intriplicate in 96 well plates and grown to confluence. Ad3 fiber knobs ormonoclonal antibodies (5 μg/ml) were added to the inner chamber. Onehour later, trastuzumab (Genentech, San Francisco, Calif.) (15 μg/ml)was added and cell viability was measured 2 hours later by WST-1 assay(Roche, San Francisco, Calif.). Three independent studies wereperformed.

Immunofluorescence analyses. Cells were cultured in 8 chamber glassslides (BD Falcon), washed twice with ice-cold PBS and then fixed withmethanol/aceton (1:1 vol/vol) for 15 min at 4° C. or with 4%paraformaldehyde for 30 min at 4° C. After fixation, cells were washedwith PBS twice and blocked with 500 μl PBS/2% dry-milk powder for 20 minat room temperature. Antibody staining was performed in 100 μl PBS for90 min at 37° C. or 4° C. overnight. If needed, secondary antibodiesdirected against the appropriate host, were applied after 3 washes withPBS for 45 min at room temperature. After 3 washes with PBS, glassslides were mounted using VECTASHIELD with DAPI (Vector Labs).Photographs were taken with a Leica DFC300FX digital camera. Confocalimages were taken on a Zeiss META confocal microscope using 40× or 100×oil lenses and Zeiss 510 software (Zeiss Microlmaging, Thornwood, N.Y.).

Electron microscopy. Polarized cells in Transwell chambers were fixedwith half-strength Karnovsky's fixative (2% paraformaldehyde, 2.5%glutaraldehyde, 0.2 M Cacodylate buffer) for one hour at roomtemperature. The fixative in the inner chamber contained 0.2% rutheniumred. The ruthenium red (Ruthenium(III) chloride oxide, ammoniated), waspurchased from Alfa Aesar (Ward Hill, Mass.). Post-fixation wasperformed with 1% OsO₄-phosphate buffer. The membranes were then cut outfrom the Transwell chambers and embedded in Medcast (Ted Pella, Redding,Calif.). Ultrathin sections were stained with uranyl acetate and leadcitrate. Processed grids were evaluated with a JEOL JEM1200EXIItransmission electron microscope. Images were acquired with an OlympusSIS Morada Digital CCD camera using ITEM software for image processing.

siRNA studies. A set of DSG2 specific siRNA was synthesized by Dharmacon(Thermo Scientific). The target sequences were CAAUAUACCUGUAGUAGAA (SEQID NO: 29), GAGAGGAUCUGUCCAAGAA (SEQ ID NO: 30), CCUUAGAGCUACGCAUUAA(SEQ ID NO: 31) and CCAGUGUUCUACCUAAAUA (SEQ ID NO: 32). Control siRNAwas purchased from Qiagen, Valencia, Calif. siRNA transfection wasperformed using HyperFect transfection reagent (Qiagen). A total of1×10⁵ HeLa were transfected with 1 ug DSG2 siRNA or control siRNA. Fortyeight hours after siRNA transfection, cells were collected with versene,and the attachment of ³H-Ad3 or ³H-Ad35 virus was analyzed as describedabove. Forty eight hours after siRNA transfection, cells were infectedwith Ad vectors at an MOI of 50 pfu/cells, and GFP expression wasanalyzed 18 hours later.

Surface plasmon resonance (SPR) analyses. Acquisitions were done on aBIAcore X instrument. HBS-N (GE-Healthcare, Pittsburgh, Pa.)supplemented with 2 mM CaCl₂ was used as running buffer in allexperiments at a flow rate of 5 μl/min. Immobilization on CM4 sensorchip(BIAcore) was performed using DSG2 (Leinco Technology, Inc.) at 0.1μg/ml diluted in 10 mM sodium acetate buffer pH4.2 injected for 10minutes on EDC-NHS activated flow-cell. A control flow-cell wasactivated by ethyl(dimethylaminopropyl) carbodiimide(EDC)/N-Hydroxysuccinimide (NHS) and inactivated by ethanolamine.Different concentration of Ad3 fiber knobs or PtDds were injected for 5minutes association followed by 3 minutes dissociation time, and thesignal was automatically subtracted from the background of theethanolamine deactivated EDC-NHS flow cell.

For experiments using biotinylated ligands, two flow cells of a CM4sensorchips were activated as described above and then coated byinjection of streptavidin (0.1 μg/ml in acetate buffer pH4.1) for 5minutes. Biotinylated ligands were then injected at 0.1 μg/ml in runningbuffer for 5 minutes on one of these two flow cells, the other beingused for background subtraction during the run. Different concentrationsof soluble DSG2 were then injected in running buffer on these flowcellsand background was automatically subtracted.

Negative stain electron microscopy. Recombinant fiber knob proteins werevisualized by negative-stain EM to assess their assembly status. Thestandard mica/carbone preparation was used with protein at 0.1 mg/ml.Samples were stained using 1% (w/v) sodium silicotungstate (pH 7.0) andvisualized on Philips CM12 electron microscope at 100 kV.

Statistical analysis: All results are expressed as mean+/−SD. Wilcoxonsigned-rank test was applied when applicable. A p-value<0.05 wasconsidered significant.

Results

Chimeric Ad5 vectors containing Ad3 fibers use DSG2 as a receptor. Ourpreliminary studies indicated that DSG2 interacting domain(s) within Ad3are formed by the fiber or fiber/penton only in the spatialconstellation that is present in viral particles, i.e. Ad3 virions orPtDds. To assess a potential role of Ad3 penton (which is present inPtDds) in binding to DSG2, we generated an Ad vector that contained Ad3fibers (Ad5/3S-GFP), but had all other capsid proteins (including thepenton) derived from Ad5. To evaluate whether the Ad3 fiber shaft had acucial role in Ad3-DSG2 interaction, e.g., contained DSG2 additionalbinding sites, we also generated a chimeric Ad5/3 vector that had theAd3 shaft substituted by the Ad5 shaft (Ad5/3L-GFP) (FIG. 9A). Notably,while the Ad3 fiber shaft contains 6 shaft repeat motifs, the Ad5 shaftis longer and contains 22 shaft motifs. For comparison, we used an Ad3vector (Ad3-GFP) containing the same GFP expression cassette as theAd5/3 vectors (33). We analyzed whether Ad5/3S-GFP and Ad5/3L-GFPvectors use DSG2 for infection. Attachment of ³H-labeled Ad vectors toHeLa cells was blocked by recombinant DSG2 protein to the same degreefor Ad3-GFP, Ad5/3S-GFP, and Ad3/5L-GFP (FIG. 9B). As expected,recombinant DSG2 also blocked transduction of all three vectors asmeasured based on GFP intentsity 18 hours after infection of HeLa cells(FIG. 9C). PtDd, used as a competitor for DSG2 interaction domainswithin the Ad virions, blocked Ad3-GFP, Ad5/3S-GFP, and Ad3/5L-GFPtransduction to similar levels (FIG. 9D). To prove the crucial role ofDSG2 in the infection of Ad3 and Ad5/3 vectors, we transfected HeLacells with siRNA specific to DSG2 mRNA or control siRNA. The meanfluorescence DSG2 intensity at 48 hours after transfection of siRNA was22.1 and 195 for DSG2 siRNA and control siRNA transfected HeLa cells,respectively, indicating efficient inhibition of DSG2 expression by DSG2siRNA. DSG2 mRNA knockdown significantly decreased Ad3-GFP, Ad5/3S-GFP,and Ad3/5L-GFP transduction (p<0.001) (FIG. 9E). Interestingly, theknockdown of DSG2 decreased Ad5/3L-GFP transduction to a lesser degree(p<0.01) than the transduction of the vectors containing Ad3 fibers(Ad3-GFP and Ad5/3S-GFP). We speculate that Ad5/3L-GFP can use receptorsother than DSG2. Taken together, these studies show: i) Ad5/3 vectorsuse DSG2 as a receptor. This has relevance for clincal studies becauseAd5/3 vectors are used in patients (14, 35) and ii) the DSG2 interactingdomains of Ad3 are located within the fiber. It appears that the Adpentons (within PtDds or Ad5 and Ad3 virions) merely provides a scaffoldfor the correct spatial constellation of Ad3 fiber knobs for interactionwith DSG2.

Crosslinking of Ad3 fiber knobs is required for efficient binding toDSG2. We then focused our attention on the Ad3 fiber. We produced in E.coli a series of recombinant Ad3 fiber knob proteins, containing thefiber knob and increasing numbers of Ad3 shaft repeats (from one to sixrepeats) (FIG. 10A). Western blot analyses using DSG2 or antiAd3 fiberknob antibodies showed that all recombinant fiber knobs formed trimers(FIGS. 10B,C). As observed previously (33), the Ad3 fiber knob plus oneshaft domain (S/Kn) did not bind DSG2 in Western blot analyses,indicating a potential steric influence of the shaft motif on the Ad3knob conformation. The protein containing 6 shaft motifs (S6/Kn) tendedto form aggregates and was therefore not used in further studies. Whenused in competition studies, all recombinant fiber knob proteinsinhibited Ad3-GFP transduction significantly less than PtDds (FIG.10D,E). We then attempted to test whether Ad3 fiber knob dimerizationwould increase DSG2 binding. Because all recombinant fiber knobscontained an N-terminal His tag (used for protein purification), wemixed Ad3 fiber knobs with antibodies against the His tag to achievetheir crosslinking. Formation of complexes between anti-His tagantibodies and fiber knob was demonstrated by electrophoresis in nativepolyacrylamide gels (FIG. 10F). When anti-His antibodies crosslinkedfiber knobs were used as competitors, a significant inhibition ofAd3-GFP transduction (compared to fiber knobs mixed with control IgG)was observed (FIG. 10G), suggesting that dimers of Ad3 fiber knobs arerequired for DSG2 binding. This appeared to be a new Ad binding strategyunique to Ad3, because anti-His antibody crosslinking of the Ad35 fiberknobs had no effect on infection by the CD46-interacting vector Ad35-GFP(FIG. 10H).

Ad3 fiber knob dimers block Ad3 infection. Crosslinking with antibodiesenhanced the blocking effect of Ad3 fiber knobs containing fewer shaftmotifs than the wild-type Ad3 fiber knob. For a potential therapeuticapplication of Ad3 fiber knobs as junction openers, we focused ourfurther studies on decreasing the size of the molecule by evaluatingfiber knob variants with the minimum number of shaft motifs, i.e. S2/Kn.Based on the finding that fiber knob cross-linking increased binding toDSG2, we generated dimers of S2/Kn by incorporating dimerizationdomains. To avoid the spontaneous fiber knob dimerization and potentialformation of inclusion bodies during production in E. coli, we utilizeda hetero-dimeric system consisting of E-coil and K-coil peptides, whichinteract with each other with high affinity (17). Two fiber knobvariants containing five repeats of EVSALEK (SEQ ID NO:22) (K-coil) andKVSALKE (SEQ ID NO:23) (E-coil) respectively, a G/S rich-flexibilitydomain followed by two shaft motifs and the homotrimeric fiber knobdomain were generated (FIG. 11A). Ad3-K/S2/Kn and Ad3-E/S2/Kn wereproduced separately in E. coli and purified by affinity chromatography.For dimerization, both purified proteins were mixed at a 1:1concentration ratio. The mixture of Ad3-K/S2/Kn and Ad3-E/S2/Kn blockedAd3 infection as efficiently as PtDd (FIG. 11B). Interestingly,Ad3-K/S2/Kn alone had the same competing strength as the mixture of bothpeptides, while Ad3-E/S2/Kn alone only inefficiently blocked infection.This suggests that Ad3-K/S2/Kn is able to homodimerize, whileAd3-E/S2/Kn is not. In support of this, we found that furthercrosslinking with anti-His antibodies increased the blocking effect ofAd3-E/S2/Kn (p<0.05), but not that of Ad3-K/S2/Kn (FIG. 11C).

Binding of a minimal dimeric Ad3 fiber knob protein to DSG2. We thenattempted to produce the smallest Ad3 fiber knob dimer, containing theK-coil or E-coil dimerization domain, only one shaft motif, and thehomotrimeric Ad3 fiber knob (Ad3-K/S/Kn and Ad3-E/S/Kn) (FIG. 12A).Because of their smaller size, such proteins have potential therapeuticadvantages in egress from blood vessels, tissue penetration and,theoretically, also contain fewer immunogenic epitopes. Ad3-K/S/Kn andAd3-E/S/Kn were produced in E. coli and purified by affinitychromatography. Analysis by polyacrylamide gel electrophoresis showedthat the vast majority of Ad3-K/S/Kn and Ad3-E/S/Kn were present astrimers (˜65-70 kDa) (FIG. 12B). Ad3-K/S/Kn alone and in combinationwith Ad3-E/S/Kn were analyzed by negative stain electron microscopy toassess their assembly status (FIG. 12C). Dimers of fiber knobs andaggregates thereof were found for both Ad3-K/S/Kn andAd3-K/S/Kn+Ad3-E/S/Kn, but larger aggregates were less abundant inAd3-K/S/Kn preparations. Both Ad3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Knblocked Ad3 attachment to HeLa cells at a level comparable to PtDd (FIG.12D). Pre-incubation of HeLa cells with Ad3-K/S/Kn andAd3-K/S/Kn+Ad3-E/S/Kn did not affect Ad5 attachment (FIG. 12E). Asexpected, Ad3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Kn also efficientlyinhibited Ad3 infection (FIGS. 12F,G). A side-by-side comparison of thefiber knobs with two and one shaft motif did not reveal significantdifferences in their ability to block Ad3 infection (FIGS. 12H,I).

SPR analysis of dimeric Ad3 fiber knob binding to DSG2. To study theinteraction of Ad3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Kn with DSG2 in moredetail, we performed surface plasmon resonance (SPR) studies. Weinitially designed binding experiments, in which DSG2 molecules wereallowed to bind to immobilized fiber knobs (FIG. 13A). Forimmobilization, fiber knobs were biotinylated and linked viastreptavidin to sensorchips. Kinetics analyses showed that bothAd3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Kn similarly recognized DSG2 with alow dissociation at the end of injection. Clearly, the binding ofsoluble DSG2 to fibers only poorly mimics the physiological interactionbetween a cell surface and the virus. We therefore immobilized thereceptor, DSG2, at the sensorchip surface and injected Ad3-K/S/Kn andAd3-K/S/Kn+Ad3-E/S/Kn, and, for comparison, PtDd and (monomeric) Ad3fiber knob at concentrations that give a similar SPR response (FIG.13B). The outcome of these studies should depend on the valence of thefiber knobs, which is trimeric for Ad3 fiber knob (monomer), 2× trimericfor Ad3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Kn, 12× trimeric for PtDd. It isfurther complicated by the fact that within PtDd not all fibers cansimultaneously interact with DSG2. While the association of the fiberknob dimers was similar (FIG. 13C “Binding at the end of association”),there were clear differences in the dissociation behavior. Ad3 fiberknob (non-dimerizing, described previously (33)) (“Ad3 knob”)dissociated faster than the other three ligands. Almost no dissociationwas seen for PtDd and Ad3-K/S/Kn+Ad3-E/S/Kn. Ad3-K/S/Kn dissociation wasbetween that of Ad3 fiber knob and Ad3-K/S/Kn+Ad3-E/S/Kn. Althoughcomplex, these data clearly show that dimeric Ad3-K/S/Kn andAd3-K/S/Kn+Ad3-E/S/Kn dissociate slower from DSG2 than does Ad3 fiberknob. This can be explained by an avidity mechanism, implying thatAd3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Kn bind to several DSG2 molecules; amechanism that allows achieving an overall low dissociation rate andhighly stable attachment. Notably, although it is possible that alldimers in Ad3-K/S/Kn+Ad3-E/S/Kn are formed by Ad3-K/S/Kn, differences indissociation rates argue against it. Further studies are required toprove this in detail.

Interaction of Ad3 with several DSG2 molecules is supported byimmunofluorescence analyses of epithelial cells (FIGS. 13D and E). Thesestudies, using Cy3-labelled Ad3 virions, suggest that one virionclusters several DSG2 proteins around itself. As outlined later, wehypothesize that this specific clustering of receptors has functionalconsequences with regards to triggering intracellular signaling andopening of epithelial junctions. Notably, in Example 1 we showed thatPtDd binding to DSG2 triggers an epithelial-to-mesenchymal transition(EMT) in epithelial cells resulting in transient opening ofintercellular junctions.

Multimeric DSG2 ligands (Ad3 virions, PtDds, Ad3-K/S/Kn) trigger openingof epithelial junctions. Epithelial cells maintain several intercellularjunctions (tight junctions, adherens junctions, gap junctions, anddesmosomes), a feature which is often conserved in epithelial cancers insitu and in cancer cell lines (29). FIG. 14A shows confocalimmunofluorescence microscopy images of polarized colon carcinoma T84cells. Shown are the cells from the lateral side, i.e. stackedXZ-layers. Intercellular junctions are visible as long vertical streaksmarked by the adhesion junction protein Claudin 7 and the desmosomalprotein DSG2. DSG2 (green) is localized at the apical end of Claudin 7signals. The tight junction protein ZO-1 can be found further apical ofDSG2 (lower panel). The latter is also visualized in XY images, whichshow a “chicken-wire” network of tight junctions marked by ZO-1 at theapical cell surface, whereas a section 1 μm deeper shows DSG2 staining(FIG. 14B). Importantly, exposure of T84 cells to Ad3-K/S/Kn triggeredpartial dissolution of epithelial junctions, reflected in decreasedstaining for DSG2 and ZO-1 (FIG. 14C), in comparison to untreated cells(FIG. 14A, lower panel).

Opening of epithelial junctions by Ad3-K/S/Kn was further confirmed byelectron microscopy (EM) studies. EM images of untreated epithelialcells show intact tight and desmosomal junctions as judged by theexclusion of the apically applied dye ruthenium red from basolateralspace (FIG. 14D, left panel). The dye appears as an electron-dense linealong the cell membrane surface. Incubation of epithelial cells withAd3-K/S/Kn resulted in leakage of ruthenium red deep into the lateralspace within 1 hour of Ad3-K/S/Kn addition (FIG. 14D right panel).Partial disassembly of desmosomes (marked by arrows) in Ad3-K/S/Kntreated cells is clearly visible in FIG. 14E. In addition to Ad3-K/S/Kn,opening of epithelial junctions was observed in confocalimmunofluorescence and EM studies with Ad3 virions, PtDd, andAd3-K/S/Kn+Ad3-E/S/Kn (data not shown). Exposure of cells to Ad3 fiberknob (non-dimerizing) or Ad3-E/S/Kn, i.e. DSG2 ligands that are unableto multimerize, had no effect on epithelial junctions (data not shown).These studies indicate that opening of epithelial junctions requiresdimers or multimers of Ad3 fiber knobs.

In addition to leakage studies with ruthenium red, we used threefunctional assays to demonstrate opening of epithelial junctions byAd3-K/S/Kn: i) Exposure of polarized epithelial cells to Ad3-K/S/Knincreased the transepithelial permeability within 30 minutes, as shownby ¹⁴C-PEG-4000 transflux studies (FIG. 15A). Importantly, permeabilityafter incubation with non-dimerizing Ad3-E/S/Kn fiber knobs or mAbsagainst different regions of the extracellular domain of DSG2was >5-fold lower than after incubation with Ad3-K/S/Kn. ii) Previousstudies showed that in polarized breast cancer BT474-M1 cells, Her2/neu,the target for Herceptin/trastuzumab, is trapped in epithelialjunctions, and that incubation of BT474-M1 cells with Ad3 PtDd increasesaccess to Her2/neu and increases trastuzumab killing of cancer cells(33). Here we used this assay to study the effect of additional DSG2ligands on trastuzumab cytotoxicity (FIG. 15B). We found that Ad3-K/S/Knsignificantly increased killing of 8T474-M1 cells by trastuzumab. Incontrast, DSG2 ligands that are not able to dimerize, i.e. Ad3-E/S/Knand a series of anti-DSG2 antibodies, had no significant effect ontrastuzumab killing. Notably, one of the anti-DSG2 antibodies againstthe extracellular domains 3/4 (mAb 6D8), appeared to stimulate tumorcell proliferation. iii) CAR, the receptor for Ad5, is localized intight junctions of polarized T84 epithelial cells (3). This is shown byconfocal immunofluorescence microscopy in T84 cells (FIG. 15C, upperpanel and FIG. 15D, upper panel). Incubation of these cells withAd3-K/S/Kn greatly increased CAR staining, which now appeared along thelateral membranes (FIG. 15C, lower panel) and at the cell surface (FIG.15D, lower panel). We speculated that this is the result of disassemblyof tight junctions and better accessibility of CAR to anti-CARantibodies that were applied to the apical side of T84 cells. Anotherpotential assay for disruption of tight junctions and CAR accessibilityis transduction with a CAR-targeting Ad vector. Infection of polarizedT84 cells (apical side) with Ad5-GFP at an MOI of 250 pfu/cell resultedin transduction of 8 (+/−2) % of cells (based on GFP-positive cellscounted 20 hours after infection) (FIG. 15E). Ad5-GFP infection in thepresence of Ad3-K/S/Kn yielded 38(+/−9) % of GFP-positive cells. Ad3-GFPin the presence of dilution buffer or Ad3-K/S/Kn transduced 17 (+/−6) %or 68 (+/−17) % of T84 cells, respectively. The latter in agreement withan earlier study, showing that Ad3 infects polarized epithelial cellsmore efficiently than Ad5 (28). This is most likely due to its abilityto bind to DSG2 and trigger junction opening. Ad3-K/S/Kn increasedAd3-GFP transduction. We speculate that the relatively small Ad3-K/S/Knprotein and it high concentration initially reaches more DSG2 receptorsthan Ad3 virions and thus enhances opening of tight junctions. In allsettings shown in FIG. 15E, most cells at the periphery of themonolayers (i.e. cells that are in contact with the walls of the innerchamber, without tight junctions) were GFP-positive. Therefore, wecounted GFP-positive cells in fields at the center of transwells. Thelatter has to be considered in the interpretation of flow cytometryanalysis of GFP expression after Ad5-GFP infection in the presence ofdilution buffer or Ad3-K/S/Kn (FIG. 15F). Although background GFPfluorescence levels were relatively high, the presence of Ad3-K/S/Kn,but not the presence of Ad3-E/S/Kn or anti-DSG2 mAbs significantlyincreased GFP expression levels after Ad5-GFP infection.

Overall, our functional studies showed that Ad3-K/S/Kn can triggeropening of epithelial junctions, while ligands that are unable tomultimerize had no effect on junctions.

Discussion

In this study, we describe two findings: i) multimerization of Ad3 fiberknob is required to achieve high-affinity and stable binding to DSG2 andii) the multimeric mode of Ad3-fiber knob/DSG2 interaction triggersopening of epithelial junctions.

Most Ad infections, including infection by the CAR-interacting Ad5, theDSG2-interacting Ad3, and the CD46-interacting Ad35 (22) target theairway epithelium. Achieving a high avidity binding to receptors with alow dissociation rate appears to be crucial for Ad infection in order tomaintain contact between virus and target cells, and more importantly,to trigger subsequent events that allow the virus to disrupt theepithelial barrier, enter the target cells, and spread within the targettissue. For CAR- and CD46-binding Ads, high avidity binding is achievedthrough interaction between the trimeric fiber knob and three receptorunits. Furthermore, as shown for the interaction between Ad11 and CD46,initial Ad binding to the receptor can trigger conformational changes inthe receptor to stabilize the binding (21); although it remains to beshown that the latter mechanism is used by Ads other than Ad11. For CAR-and CD46-interacting Ads, attachment involves the fiber knob and thereceptor, and it can be completely blocked by an excess of soluble fiberknob. This is not the case for the DSG2-binding Ad3. We have shown thatcomplete inhibition of Ad3 binding and infection requires the physicallinkage and, most likely, a specific spatial constellation of at leasttwo fiber knobs. This is achieved with Ad virions, Ad3 PtDds or dimericAd3-K/S/Kn. These ligands appear to achieve simultaneous binding toseveral DSG2 molecules, which, on the one hand, provides a high avidityand, on the other hand, is functionally relevant for opening ofepithelial junctions. We are currently conducting crystal structure andmutagenesis studies to further support our findings on Ad3-DSG2interaction.

In order to initiate infection, many pathogens have evolved mechanismsto disrupt junctional integrity. Vibrio cholera strains produce Zonaoccludens toxin (Zot), which possess the ability to reversibly alterintestinal epithelial junctions, allowing the passage of macromoleculesthrough mucosal barriers (6). Clostridium perfringens enterotoxinremoves claudins-3 and -4 from the tight junctions to facilitatebacterial invasion (25). Furthermore, oncoproteins encoded by human Ad,HPV, HTLV-1 can transiently open epithelial junctions by mislocalizingthe junction protein ZO-1 (15). The latter mechanisms used by virusesappear to play a role in lateral viral spread. However, for an efficientinitial infection, attachment of virus must be linked with triggeringthe opening of epithelial junctions.

In Example 1, using immunofluorescence, PI3K/MAPK phosphorylation, mRNAexpression array, and metabolic pathway inhibition approaches, wereported that binding of DSG2 by Ad3 virions and Ad3 PtDds triggers anepithelial-to-mesenchymal transition in epithelial cells, resulting intransient opening of intercellular junctions. Intercellular junctionopening mediated by interaction of Ad3 particles or recombinant PtDdswith DSG2 was further supported by increased epithelial cellpermeability and access to receptors that are trapped in intercellularjunctions (e.g. Her2/neu). In the present study, we providemorphological (confocal immunofluorescence, EM) and functional(permeability, trastuzumab killing, Ad5 infection) data showing thatAd3-K/S/Kn also triggers junction opening. Importantly, other DSG2ligands that are not able to multimerize and cluster DSG2 such asmonomeric Ad3-E/S/Kn or monoclonal antibodies against different regionsof DSG2 were unable to efficiently open junctions. It remains, however,the question how Ad3 can pass the tight junctions that are located atthe apical side of DSG2/desmosomes. We speculate that efficientinfection occurs through a positive feed-forward mechanism because theAd-DSG2 mediated junction opening occurs within minutes thereby exposingDSG2 trapped in junctions to viral particles that are present at thesite of infection.

In the discussion of Ad3 infection mechanisms, it is also noteworthythat, during Ad3 replication, PtDds are formed at a massive excess of5.5×10⁶ PtDds per infectious virus (7). This suggests that PtDdformation is functionally important for Ad3, i.e. advantageous in spreador persistence of Ad3. A similar mechanism appears to take place ininfection of epithelial cells by Ad5. During replication of Ad5, excessproduction of fiber results in the disruption of epithelial junctionseither by interfering with CAR dimerization or by triggeringintracellular signaling that leads to reorganization of intercellularjunctions (4, 32). Recently, it has been suggested (based on in vitrostudies) that CD46 is exposed apically on polarized epithelialrespiratory cells and is therefore more likely to function as an Adreceptor for initial infection in vivo (9). We could not confirm this inhuman CD46 transgenic mice, where the CD46 expression pattern did notcorrelate with Ad transduction (19). Moreover, in polarized epithelialcancer cultures, we found CD46 trapped in intercellular junctions andnot accessible to Ad35 applied to the cell surface (33). At this point,we believe that the question of how CD46-binding Ads disrupt theepithelial barrier remains to be answered.

The finding that Ad3-K/S/Kn triggers junction opening has practicalimplications because intercellular junctions represent physicalobstacles for access and intratumoral dissemination of anti-cancertherapeutics (26-27). An epithelial junction opener would be relevantfor cancer therapies with monoclonal antibodies directed againsttumor-associated antigens that are trapped in epithelial junctions (e.g.Her2/neu or EGFR1) (33). A junction opener might also improve theefficacy of adoptive T-cell therapy (5) or treatment with liposomalchemotherapy drugs (10). Finally, Ad3-K/S/Kn might increase thatefficacy of transduction of normal or malignant tissues by Ad5-basedgene therapy vectors. The biotechnological applicability of Ad3-K/S/Knis further underscored by the ease of its production and purificationand by the fact that it spontaneously homodimerizes.

In conclusion, our study sheds light on the mechanisms of Ad3 infectionof epithelial cells. The finding that Ad3-K/S/Kn, a small recombinantprotein, triggers opening of epithelial junctions has implications forcancer therapy and drug delivery into epithelial tissues.

References for Example 2

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Example 3 A Recombinant Epithelial Junction Opener Improves MonoclonalAntibody Therapy of Cancer Abstract

The therapeutic efficacy of monoclonal antibodies (mAbs) for cancertreatment is limited by intercellular junctions that tightly linkepithelial tumor cells to one another. We generated a small, recombinantadenovirus serotype 3 derived protein, junction opener 1 (JO-1), whichbinds to the epithelial junction protein desmoglein 2 (DSG2). Wedemonstrate in mouse models with tumors derived from Her2/neu- andEGFR-positive human cancer cell lines that within one hour afterintravenous injection, JO-1 mediates the cleavage of DSG2 dimers inintercellular junctions as well as the activation of intracellularsignaling pathways that lead to a decrease in the level of the tightjunction protein E-cadherin. JO-1-triggered changes in epithelialjunctions enabled better intratumoral penetration of the anti-Her2/neumAb trastuzumab (Herceptin) as well as for improved access to its targetreceptor, Her2/neu, which is partly trapped in junctions. This directlytranslated in a better therapeutic efficacy of trastuzumab in xenograftmodels using Her2/neu-positive breast, gastric, and ovarian cancercells. Furthermore, the combination of JO-1 with the EGFR-targeting mAbcetuximab (Erbitux) greatly improved the therapeutic outcome in a modelof metastatic EGFR-positive lung cancer. A combination ofJO-1/trastuzumab treatment with an additional approach that allows forthe transient degradation of tumor stroma proteins resulted in completetumor eradication. In addition to its clinical relevance for cancertreatment, this study also sheds light on the mechanism of adenovirusserotype 3 infection of epithelial cells.

Introduction

Monoclonal antibodies (mAbs) have emerged as a class of novel oncologytherapeutics. To date, there are 21 marketed therapeutic mAbs for thetreatment of cancer, with hundreds of more currently in clinicaldevelopment. Among the marketed mAbs are trastuzumab (Herceptin) andcetuximab (Erbitux). Trastuzumab targets the human epidermal growthfactor receptor 2 (Her2/ErbB-2). The receptor for cetuximab is the humanepidermal growth factor receptor 1 (Her1/ErbB-1). Both receptors belongto the family of tyrosine kinase receptors and initiate signalingthrough several pathways which promote cell survival and proliferation(Harari et al., 2007). Trastuzumab is used as a first line therapy inHer2/neu positive breast cancer patients and has also been approved formetastatic Her2/neu positive gastric cancer. Current FDA-approvedindications for cetuximab include colorectal, head and neck, lung, andpancreatic cancer (Wheeler et al., 2010). Most patients with early stagebreast or colon cancer have a measurable tumor response to trastuzumaband cetuximab therapy. However, in patients with advanced or recurrentdisease, the response rate to these mAbs is only 8% to 10% (Adams andWeiner, 2005).

The mechanisms of trastuzumab and cetuximab action include theactivation of antibody-dependent or complement-dependent cytotoxicity,and interference with tyrosine kinase receptor signaling that isrequired for tumor cell survival (Wheeler et al., 2010). A unifyingaspect among these mechanisms is that tumor cell growth inhibition isdependent on the binding of mAbs to their corresponding receptors.Therefore, molecules that prevent access and binding to the receptor,either by physically inhibiting intratumoral transport from bloodvessels to malignant cells or masking of receptors, are predicted toblock trastuzumab and cetuximab activity (Lesniak et al., 2009). Amongthese molecules are tumor stroma proteins such as collagen or laminin(Li et al., 2004). In a recent study, we demonstrated that transientdegradation of these stroma proteins significantly improved trastuzumabtherapy (Beyer et al., 2011).

In addition to obstacles formed by tumor stroma proteins, the epithelialphenotype of cancer cells also creates physical barriers to cancertherapy (Strauss and Lieber, 2009; Strauss et al., 2009). Severalstudies demonstrated that the expression or upregulation of epithelialproteins correlated with increased resistance to trastuzumab (Fessler etal., 2009) and cetuximab (Oliveras-Ferraros et al., 2011) therapy ofbreast and colorectal cancer, respectively. Epithelial cells maintainseveral intercellular junctions (tight junctions, adherens junctions,gap junctions, and desmosomes), a feature which is often conserved inepithelial cancers in situ and in cancer cell lines (Turley et al.,2008). Epithelial junctions are composed of adhesive dimers consistingof cadherin molecules derived from two neighboring cells (Koeser et al.,2003). Desmogleins 1, 2, and 3 (DSG1-3) and desmocollins 1, 2, and 3(DSC1-3) are subclasses of cadherins. DSC2 and DSG2 are widely expressedand are found together in desmosomes of the basal layer of epithelialcells. The cytoplasmic tails of the desmosomal cadherins link the plasmamembrane to the cytoskeleton through a complex of proteins, whichinclude plakoglobin, desmoplakin, and plakophilins. Desmoglein 2 (DSG2)is overexpressed in a series of epithelial malignancies, includingbreast cancer (Wang et al., 2011b) (Suppl. FIG. 1), ovarian cancer (Wanget al., 2011b) (Suppl. FIG. 1), lung cancer (Wang et al., 2011b),gastric cancer (Biedermann et al., 2005), squamous cell carcinomas(Harada et al., 1996), melanoma (Schmitt et al., 2007), metastaticprostate cancer (Trojan et al., 2005), and bladder cancer (Abbod et al.,2009).

In Example 1, we demonstrated that a group of human adenoviruses (Ads)(Ad serotype 3, 7, 11, and 14) use DSG2 as a primary attachment receptorfor the infection of cells. Importantly, in epithelial cells, Ad3binding to DSG2 triggered activation of signaling pathways resulting inthe transient opening of epithelial junctions. The opening of junctionswas reflected by an increased transepithelial permeability and by theunmasking of proteins that are trapped in tight junctions, e.g., thecoxsackie-adenovirus receptor or the zonula occludens-1 protein. Theopening of the epithelial junctions was also achieved with recombinantsubviral particles, such as the penton-dodecahedra (PtDd), consisting of12 Ad3 fibers linked to their penton bases (Fuschiotti et al., 2006;Norrby et al., 1967). We subsequently generated a minimal Ad3-derivedDSG2 ligand formed by two fiber knob domains. This protein, with amolecular weight of approximately 50 kDa, is produced in E. coli and canbe easily purified. In a series of functional studies we demonstratedthat this protein efficiently triggers the opening of the junction. Inthe following study, we therefore refer to this protein as junctionopener-1 (JO-1).

In this study, we have partially delineated the in vivo mechanism ofJO-1-mediated junction opening. We show that Her2/neu and EGFR aretrapped in the intercellular junctions in xenograft tumors. JO-1treatment greatly increased the permeation of mAbs in tumors andsignificantly increased the efficacy of trastuzumab and cetuximabtherapy in a series of xenograft tumor models.

Results

JO-1 triggers opening of epithelial junctions. In the examples above, weshowed that Ad3 particles or recombinant Ad3 penton-dodecahedra (PtDd)(FIG. 16A) bind to DSG2 and trigger the transient opening of epithelialjunctions, which, in turn, increases the killing of Her2/neu positiveBT474 breast cancer cells by trastuzumab. As the large size of Ad3 orPtDd particles can affect their egress from blood vessels and tissuepenetration, we generated smaller Ad3-derived DSG2 ligands that arefunctionally active as epithelial junction openers. We found thathigh-affinity binding to DSG2 and subsequent junction opening requiresAd3 fiber dimers. Based on this finding, we designed a small,self-dimerizing Ad3 fiber derivative, called junction-opener 1 (JO-1)(FIGS. 16A, B). JO-1 has a molecular weight of ˜50 kDa and is producedin E. coli prior to purification by affinity chromatography.

The functional activity of JO-1 was tested on polarized colon cancer T84cells. This demonstrated that incubation of cells with JO-1 triggeredremodeling of epithelial junctions, as shown by confocal microscopy forClaudin 7 and DSG2 (FIG. 16C). The desmosomal protein DSG2 is overlapswith adherens junctions marked by Claudin 7. Within 30 min after JO-1binding to DSG2 Claudin 7 staining increases, which is most likely theresult of better accessibility of antibodies to Claudin 7 (which areapplied to the inner chamber of transwell cultures, i.e. the apical sideof polarized epithelial cells). Opening of the tight junctions, whichare localized apical to the desmosomal and adherence junctions, isillustrated by electron microscopy (FIG. 16D). Microphotographs ofuntreated epithelial cells show intact tight junctions as judged by theexclusion of the apically applied dye ruthenium red from basolateralspace. Incubation of epithelial cells with JO-1 for 1 hour resulted inthe disassembly of tight junctions and leakage of ruthenium red into thebasolateral space (FIG. 16D, right panel). Exposure of polarizedepithelial cells to JO-1 also increased the transepithelialpermeability, as shown by transflux of ¹⁴C-PEG-4000 with a molecularweight of 4000 Da (FIG. 16E). Importantly, monoclonal antibodies againstdifferent regions of the extracellular domain of DSG2 did notsignificantly increase transepithelial permeability. We speculate thatthe ligation of several DSG2 molecules is required to trigger theopening of the junctions. Microscopy and permeability studies show thatJO-1 triggers the opening of junctions within minutes. The effect ofJO-1 is transient as is illustrated by the fact that 60 min subsequentto the removal of a JO-1 pulse treatment, junction structure andpermeability is restored to normal morphology. When JO-1 was left oncells, the morphological changes in epithelial junctions could still beseen at 24 hours after the initial addition of JO-1 (data not shown).

JO-1 triggers intracellular signaling and increases penetration of mAbin epithelial tumors in vivo. A breast cancer xenograft model was usedto study the effect of JO-1 on epithelial junctions in vivo. Humanbreast cancer HCC1954 cells were injected into the mammary fat pad ofCB17-SCID/beige mice. The resulting tumors resembled the histology ofbreast cancer in humans (Li et al., 2004), i.e. tumors were vascularizedand contained nests of epithelial cells glued together by epithelialjunctions and surrounded by extracellular matrixes). When tumors reacheda volume of ˜200 mm³, JO-1 was injected intravenously. JO-1 could bedetected in the tumors by immunofluorescence microscopy as early as 1hour post-injection. JO-1 accumulated in the tumors as is indicated bythe increased immunofluorescence at 12 hours post injection (FIG. 17A,left three panels). This is also confirmed by Western blot analysis oftumor lysates (FIG. 17A, right panel). Analysis of DSG2 on tumorsections by immunofluorescence microscopy in PBS treated animals showedmembrane localized signals (FIG. 17B, left panel). One hour subsequentto JO-1 injection, DSG2 molecules were mostly found in the cytoplasm ofthe tumor cells (second panel). By 12 hours, membrane localization ofDSG2 appeared to be partly restored (third panel). Western blot analysisusing anti-DSG2 antibodies against the extracellular domain of DSG2revealed smaller fragments of the DSG2 extracellular domain (ECD),probably produced by proteolytic cleavage, at the 1 hour time point(FIG. 17B, right panel). Taken together these data suggest that JO-1triggers cleavage within the DSG2 ECD and DSG2 internalization. Wespeculate that this disrupts DSG2 dimers between two neighboringepithelial tumor cells and contributes to remodeling of lateraljunctions. No toxic side effects or changes in histology of normalepithelial tissues of the gastrointestinal or respiratory tracts wereobserved in JO-1 treated animals.

In the examples above, it was found in in vitro studies that Ad3 bindingto DSG2 of epithelial cells triggered intracellular signaling includingpathways that are involved in epithelial-to-mesenchymal transition(EMT). EMT is a reprogramming process involved in embryonal development,but also in tumor metastasis. Among the feature that characterize EMTare decreased expression of epithelial markers, altered location oftranscription factors, and activation of Erk1/2 (MAPK) (Turley et al.,2008). In our studies with xenograft tumors, we found lessnon-phosphorylated and phosphorylated forms of E-cadherin in tumors 12hours after intravenous injection of JO-1 (FIG. 17C, left panel).Preceding the changes in E-cadherin, was an increase in phosphorylatedErk1/2 (FIG. 17C, compare pErk1/2 PBS vs. JO-1 (1 h)). It is wellestablished that ERK1/2 activation results in a decrease of E-cadherinexpression and phosphorylation during EMT (Andarawewa et al., 2007;Larsen et al., 2003; Turley et al., 2008). The decrease in E-cadherinand an increase in signals for phosphorylated Erk1/2 upon JO-1 injectionwere also observed by immunofluorescence microscopy (FIG. 17C, rightpanels).

Overall, these studies indicate that JO-1 triggers activation of Erk1/2pathways in vivo, in HCC1954 tumors. Based on this, we hypothesize thatopening of epithelial junctions by binding of JO-1 to DSG2 involves atleast two mechanisms: i) cleavage of the DSG2 ECD, and disruption ofDSG2 dimers with subsequent internalization; and ii) induction ofEMT-like events through the activation of Erk1/2.

Next, we tested whether JO-1-triggered opening of epithelial junctionsin tumors would increase the penetration of monoclonal antibodies intoxenograft tumors. Trastuzumab, which is a humanized IgG1 mAb, wasinitially utilized for this study. As in previous studies, trastuzumabwas injected intraperitoneally at a dose of 10 mg/kg. Trastuzumab wasvisualized in tumors with antibodies specific to human IgG. In tumorsections and Western blot analyses, trastuzumab was detectable 1 hourpost-injection and at higher levels 12 hours after injection (FIG. 18).Intravenous injection of JO-1 one hour prior to the administration oftrastuzumab, visibly increased the amount of trastuzumab in the tumors,indicating either better egress from blood vessels, better intratumoralpenetration, and/or longer intratumoral half-life.

mAb targets are trapped in epithelial junctions. In breast cancerxenograft sections and in cultured breast cancer cells, we foundco-staining of Her2/neu and the adherens junction protein Claudin 7(FIG. 19A,). Confocal microscopy of breast cancer BT474 cells confirmedthe trapping of Her2/neu in lateral junctions. This is in agreement withan earlier study, demonstrating that Her2/neu is a basolateral proteinthat becomes accessible from the apical surface only when the tightjunctions are disrupted (Vermeer et al., 2003). Incubation of theHer2/neu positive breast cancer cell lines BT474 (FIG. 19) or HCC1954(not shown) with JO-1 changed the composition of the lateral epithelialjunctions within 1 hour. As a result of this, Her2/neu staining at thecells surface became more intense, while it faded in areas distal to thecell surface. This suggests that JO-1 mediated junction openingtriggered a translocation of Her2/neu from lateral membranes to the cellsurface. The effect of JO-1 on lateral junctions was transient andcellular morphology returned to that of control cells by 16 hour afterJO-1 pulse treatment. Being trapped in epithelial junctions also appearsto be a problem for other cancer therapy targets such as EGFR1 (thetarget for cetuximab/Erbitux) as co-staining for EGFR and the tightjunction protein E-cadherin suggests (FIG. 19B). In our studies withcetuximab we focused on a lung cancer model (A549 cells), as most coloncancer cell lines have mutations in K-ras, which confers resistance tocetuximab (Karamouzis et al., 2007). Similar to what we observed forHer2/neu, incubation of A549 cells with JO-1 resulted in a translocationof EGFR to the cell surface.

Release of mAb receptors from trapping is supported by the enhancedkilling of cancer cells by trastuzumab and cetuximab. In vitro killingof BT474 breast cancer and A549 lung cancer cells by trastuzumab andcetuximab, respectively, was inefficient (FIGS. 19C and D). Pretreatmentof these cells with JO-1 significantly increased in vitro cytotoxicityof both antibodies in the corresponding cell lines.

Overall, these studies indicate that JO-1 mediates junction openingthereby allowing for a better intratumoral penetration of mAbs, as wellas improving the access to their target receptors that otherwise aretrapped in the junctions. Based on this we performed a series of in vivostudies to investigate whether JO-1 pre-treatment can improve thetherapeutic efficacy of mAbs in xenograft models.

JO-1 improves trastuzumab therapy in vivo. JO-1's potential enhancementof trastuzumab therapy was first tested in an orthotopic breast cancermodel based on Her2/neu positive BT474-M1 cells. JO-1 injection alonehad no significant effect on tumor growth (FIG. 20A). BT474-M1 tumorsinitially responded well to trastuzumab; however, pre-injection of JO-1significantly enhanced the therapeutic efficacy of trastuzumab (FIG.20A). These findings are in agreement with our studies with PtDd in thismodel. The enhancing effect of JO-1 pretreatment becomes more apparentwhen treated mice were followed long-term, i.e. for 136 days. While 60%of the animals that received trastuzumab monotherapy relapsed around day100, none of the animals treated with JO-1+trastuzumab showed tumorre-growth (data not shown).

A second breast cancer model involved HCC1954 cells. Tumors derived fromthese cells are more resistant to trastuzmab (FIG. 20B). As seen in theBT474-M1 model, JO-1 pretreatment significantly improved trastuzumabtherapy and stalled tumor growth. The enhancing effect of JO-1 wascomparable to that of PtDd (data not shown). Based on our study withPtDd, we chose a time interval of 10 hours between JO-1 and trastuzumabinjections. This regimen is supported by the kinetics of JO-1accumulation in tumors and the kinetics of E-cadherin decrease (seeFIGS. 17A and C). On the other hand, events that appear to be linked tojunction opening, i.e. DSG2 cleavage or Erk1/2 activation, occur alreadywithin 1 hour after JO-1 injections. We therefore investigated howsimultaneous JO-1/trastuzumab injection and injection of trastuzumab 1hour after JO-1 application influenced the therapeutic outcome (FIG.20C). In this study no significant difference was found when compared tothe treatment approach used initially (trastuzumab 10 hours after JO-1).We speculate that this is due to the relative slow accumulation of theprotein in the tumors.

To further consolidate the clinical relevance of JO-1 as aco-therapeutic for trastuzumab, we performed efficacy studies in aHer2/neu-positive gastric cancer (NCI-N87) model (FIG. 21). Similar tothe breast cancer model, we found co-staining of Her2/neu and Claudin 7in NCI-N87 cultures and xenograft tumors, suggesting trapping ofHer2/neu in epithelial junctions. To establish the gastric cancerxenograft model, NCI-N87 cells were injected subcutaneously.Pretreatment of tumor-bearing mice with JO-1 significantly improvedtrastuzumab therapy as reflected by delayed tumor growth.

JO-1 improves cetuximab therapy in vivo. A xenograft model withsubcutaneous tumors derived from A549 lung cancer cells was initiallyutilized. Cetuximab treatment of mice with pre-established A549 tumorsdid not result in a significant delay of tumor growth when compared totreatment with PBS. JO-1 was injected intravenously or intraperitoneallyfollowed by cetuximab 12 hours later. Both treatment approaches had asignificant therapeutic effect and resulted in a decrease of tumorvolumes (FIG. 22A). An additional combination of intravenously injectedJO-1 with an intratumoral application of the junction opener did notfurther increase the therapeutic efficacy. As seen in the breast cancermodel, JO-1 treatment alone did not exert a significant anti-tumoreffect. JO-1 pretreatment enhanced cetuximab therapy to a similar degreeas seen with PtDd (FIG. 22B).

Next, the co-therapy approach was tested in an orthotopic lung cancermodel. To establish this model, A549 cells were injected intravenously.In this model, mice became morbid within 37 days of tumor celltransplantation with predominant tumor localization to the lung (FIG.22C, “PBS” group). Treatment of mice was started at day 10, i.e. 27 daysbefore the animals in the control group reached the endpoint of thestudy. All animals were sacrificed at day 40. While lung metastases wereclearly visible in the control group, JO-1 group, as well as thecetuximab treated animals, 80% of the lungs in theJO-1+cetuximab-treated animals were free of tumor when inspectedmacroscopically. Microscopy of lung sections showed that in PBS treatedanimals, tumor cells almost completely replaced normal lung tissue andalso filled the bronchioli. (FIG. 22C, right panels). Invasion into thebronchioli was less pronounced in cetuximab injected animals.Importantly, while cetuximab treated animals had considerable,infiltrating tumor growth, the majority of JO-1+cetuximab injectedanimals showed only micrometastases.

Combined tumor stroma protein degradation and junction opening. We haverecently shown in immunohistochemical studies of tumor sections fromcancer patients and xenografts that the extracellular matrix proteinsforming the tumor stroma tightly surround nests of malignant breast andcolon cancer cells (Li et al., 2009). Transient degradation of tumorstroma proteins by intratumoral expression of the peptide hormonerelaxin significantly enhanced trastuzumab therapy (Beyer et al., 2011).Here, we utilized the HCC1954 model to test whether additional transienttumor stroma protein degradation, would further increase the effect ofJO-1 on trastuzumab therapy (FIG. 23A). To deliver the relaxin gene tothe tumor we employed an approach based on hematopoietic stem cells(HSCs) (Li et al., 2009). This approach capitalizes on the observationthat tumor cells secrete a number of chemokines that actively mobilizemyeloid progenitors from the bone marrow and recruit them to the tumorstroma, where they differentiate into tumor-associated macrophages(TAMs). TAMs are critical for tumor survival as they produce factorsthat trigger/support tumor growth, neo-angiogenesis, immune escape andstroma development. The approach involved the ex vivo transduction ofbone marrow derived HSCs with lentivirus vectors expressing thetransgene under control of a Doxycycline (Dox)-inducible transcriptioncassette, and the transplantation of these cells into myelo-conditionedrecipients, where they engraft in the bone marrow and provide along-term source of genetically modified cells that will home intotumors. This study (FIG. 23B) showed that relaxin expression alonesignificantly delayed tumor growth and increased trastuzumab therapy.The combination of relaxin expression and JO-1 treatment stopped tumorgrowth. Tumors did not re-grow when treatment was terminated, incontrast to groups that received either relaxin+trastuzmab orJO-1+trastuzumab therapy. Histological analyses of residual masses inthe JO-1/relaxin/trastuzumab group at the end of the observation period,showed only connective tissue. In contrast, explanted tumors from theother groups contained tumor cells, which could be cultured in vitroupon protease digestion of tumors. Notably, no adverse side effects wereobserved in mice that received the triple combination(JO-1/relaxin/trastuzumab) treatment.

Our data underscore that physical obstacles in tumors are involved inmediating resistance to trastuzumab therapy.

Effect in the presence of antibodies against JO-1 on in vitrocytotoxicity assays. Ad3 is a widely distributed pathogen. As such, JO-1is a viral protein, which can potentially be immunogenic, and caninterfere with its therapeutic activity after repeated administration.Ten out of 30 serum samples from breast cancer patients tested positivefor neutralizing Ad3 antibodies (data not shown). This is in agreementwith reports that the serum prevalence for neutralizing anti-Ad3antibodies in humans (by age 10) is ˜40% (Sakamoto et al., 1995). Weshow, however, that the anti-Ad3 antibody-positive human sera did notreact with JO-1 (data not shown). This is not surprising as mostneutralizing antibodies are directed against the Ad hexon (Sumida etal., 2005). To obtain anti-JO-1 positive serum, mice were vaccinatedwith JO-1 (data not shown). In in vitro cytotoxicity studies, nointerference was found of the anti-JO-1 antibodies with the enhancingeffect of JO-1 on trastuzumab killing of Her2/neu-positive BT474 cells(data not shown). Along this line, pooled human serum from healthyvolunteers or breast cancer patients also did not interfere with thefunction of JO-1.

Discussion

JO-1 as new co-therapeutic. Despite their success in many areas, thetherapeutic efficacy of mAbs are limited, with only a minority ofpatients responding to these agents as monotherapies. The action of manyanti-cancer mAbs, including trastuzumab and cetuximab, involve bindingto the corresponding target receptors on tumor cells. Target receptormasking or preventing intratumoral dissemination of mAbs is a potentialshielding and escape mechanism for cancer cells (Lesniak et al., 2009).In addition to the barriers formed by tumor stroma proteins, such ascollagen or laminin, the epithelial phenotype of epithelial cancer alsocreates obstacles to mAb therapy. A recent study reported that theexpression of epithelial features such as mucin 1 correlated withtrastuzumab resistance (Fessler et al., 2009). Furthermore, theupregulation of E-cadherin contributed to resistance to cetuximabtherapy of colorectal cancer (Oliveras-Ferraros et al., 2011).

While it has long been known that tumor invasion and metastasis isaccompanied by EMT, recent studies have shown that this process can bereversed by a mesenchymal-to-epithelial transition (MET) (Thiery andSleeman, 2006). Regaining epithelial features, including epithelialjunctions, appears to represent a protection mechanism for the tumorthat also shields it from anti-cancer treatment (Brennan et al., 2010;Strauss et al., 2009; Thiery and Sleeman, 2006). In this context, wefound in studies on primary ovarian cancer cells that acquisition ofchemoresistance and the formation of putative cancer stem cells involvedprocesses reminiscent of MET (Strauss et al., 2011).

We hypothesized that an approach that allows for a transient dissolutionof epithelial junctions could improve the therapeutic efficacy of mAbssuch as trastuzumab and cetuximab. In the context of basicadenovirological studies, we designed the small recombinant proteinJO-1. JO-1 binding to DSG2 resulted in the transient opening of tightjunctions. This increased the penetration of trastuzumab in the tumorand allowed for better access to mAb target receptors, which, in turn,facilitated mAb therapy in a series of xenograft models involving humanepithelial tumor cells. Potentially, the combination of JO-1 withtrastuzumab and cetuximab might allow the reduction of the effectivedose of these mAbs, thereby reducing critical side effects, i.e.trastuzumab-associated cardiotoxicity and acne-like rashes that oftenoccur during cetuximab therapy.

JO-1 is an attractive therapeutic for development because it can beproduced in high yields in E. coli. Notably, DSG2 ligands that are notable to cluster DSG2, such as monoclonal antibodies against DSG2, areinefficient in junction opening in the tumor models used in this study.To our knowledge, there is no other therapy in development that binds toDSG2, and is able to sensitize tumors to mAbs. A series of pathogenshave evolved mechanisms to disrupt junction integrity. For example,Vibrio cholerae strains produce Zona occludens toxin (Zot), whichpossesses the ability to reversibly alter intestinal epithelialjunctions, allowing the passage of macromolecules through mucosalbarriers (Fasano et al., 1991). A Zot-derived hexapeptide (AT-1001) hasbeen developed, however clinical testing in patients with coeliacdisease was recently stopped due to safety concerns and the lack ofefficacy.

Mechanisms of action. Our data suggest that JO-1 triggers junctionopening in epithelial tumors through two, potentially connectedmechanisms: the disruption of DSG2 dimers in intercellular junctionsand/or intracellular signaling that leads to a decrease of E-cadherinand potentially other junction proteins. Because cadherins form dimersbetween neighboring cells, a number of pathogens have evolved mechanismto trigger cleavage within the extracellular domain of cadherins.Candida albicans, an organism able to invade the bloodstream via thegastrointestinal tract, binds to E-cadherin and triggers cleavage ofboth intra- and extracellular domains of E-cadherin, therebydestabilizing the homotypic interactions between adjacent epithelialcells (Frank and Hostetter, 2007). Furthermore, Bacteroides fragilisenterotoxin and Porphyromonas gingivalis gingipains have been associatedwith the degradation of E-cadherin (Katz et al., 2000; Wu et al., 1998).Both of these studies attributed the cleavage of E-cadherin to aspecific bacterial protease. Our data suggest that Ad3 uses a similarmechanism to breach the epithelial barrier in the respiratory tract. Weare currently studying the mechanisms of DSG2 cleavage. In ongoingstudies, we are attempting to determine whether JO-1 binding triggersconformational changes within DSG2. Dissolution of DSG2 dimers might inturn destabilize more apically localized tight junctions as discussedrecently (Koeser et al., 2003).

Based on in vitro studies above, Ad3 binding to DSG2 triggers theactivation of Erk1/2 and other pathways involved in EMT. This studyshows that Erk1/2 is activated in tumors within 1 hour after intravenousJO-1 injection. Our finding is in agreement with previous reportsshowing that cadherins engage in bidirectional signaling with thereceptor tyrosine kinases to regulate intercellular junctions (Klessneret al., 2009). We are currently generating various DSG2 knockout modelsand truncated/mutated DSG2 variants to better delineated the role ofDSG2 in maintaining epithelial junctions.

Side effects on normal epithelial tissues. In humans and non-humanprimates, DSG2 is expressed in the gastro-intestinal and respiratorytracts (Li et al., 2011). As the mouse orthologue of DSG2 is notrecognized by Ad3 or JO-1 (Wang et al., 2011b), safety studies in normalmice are relatively inadequate. We have therefore generated transgenicmice containing the human DSG2 locus. The expression pattern and levelof human DSG2 in these animals were similar to those found in humans.Furthermore, we showed that JO-1 binding to human DSG2 in transgenicmouse epithelial cells triggered junction opening to a degree similar todata observed in human cells. In preliminary studies withDSG2-transgenic mice we did not find critical side effects ofintravenous JO-1 injection (2 mg/kg) (Li et al., 2011). We speculatethat DSG2 in normal epithelial cells is not readily accessible tointravenously applied JO-1. On the other hand, greater leakage oftumor-associated blood vessels and the lack of strict cell polarizationmight make epithelial tumors more responsive to JO-1. Lack of toxicityafter intravenous injection of JO-1 ligands is also underscored bystudies with adenoviruses containing Ad3 fibers. These viruses bind toDSG2 and act on junctions the same way as JO-1. Intravenous injection ofAd5/3 or Ad3 oncolytic vectors into humans was found to be safe(Hemminki et al., 2010; Koski et al., 2010). We are currently conductingdetailed toxicity studies in non-human primates and DSG2-transgenic miceto validate the potential of JO-1 as a safe therapeutic.

JO-1 immunogenicity. As JO-1 is a viral protein, adaptive immuneresponses might develop in humans, particularly after repeatedinjection. This might, however, not be a clinical problem. Thefirst-line treatment of Her2/neu-positive metastatic breast cancer istrastuzumab in combination with chemotherapeutic drugs (doxorubicin,cyclophosphamide, paclitaxel, or docetaxel). Also, cetuximab is used incombination with chemotherapeutics (vinorelbine plus cisplatin) innon-small cell lung cancer patients. It is likely that theimmunosuppressive chemotherapy prevents immune responses against JO-1.Again, this expectation is supported by studies with oncolyticadenovirus vectors. In these studies, immunosuppression allowed forrepeated vector application (Thomas et al., 2008). On the other hand, itwould be beneficial if JO-1 could be used in immunocompetent cancerpatients. For example, JO-1 can theoretically act as animmunostimulatory monotherapy by increasing the access and intratumoralpenetration of pre-existing or transplanted anti-tumor T-cells.Tumor-specific T-cells are frequently found in untreated breast cancerpatients, even at late stages of disease, and transfer of these cellsinto mice has been shown to reject xenotransplanted autologous tumors(Beckhove et al., 2004; Disis et al., 1994; Disis et al., 2000; Feuereret al., 2001; Toso et al., 1996). Therefore, we started to investigatein more detail JO-1 immunogenicity. Despite the fact that approximatelyone third of humans have neutralizing antibodies against Ad3, ourstudies showed these antibodies did not interact with JO-1 (Suppl. FIG.5). Furthermore, anti-JO-1 antibodies generated by vaccination of micedid not affect the enhancing effect of JO-1 on trastuzumab killing invitro. This is most likely due to the high affinity of the JO-1-DSG2interaction (Wang et al., 2011a). We are currently establishingsyngeneic tumors in hu-DSG2-transgenic mice to investigate how anti-JO-1immune responses affect its efficacy.

Potential risk to enhance tumor invasion and metastasis. In agreementwith other studies (Wang et al., 2011b), (Biedermann et al., 2005),(Harada et al., 1996), (Schmitt et al., 2007), (Trojan et al., 2005),(Abbod et al., 2009), we found a higher DSG2 expression in malignanttissues than in the surrounding normal epithelial tissue. Furthermore, arecent study on squamous cell cervical cancer demonstrated that incontrast to all other desmosomal proteins, DSG2 was up-regulated ininvasive cancer (Kurzen et al., 2003). There are, however, also studiesreporting a reduction in the amounts of DSG2 in invasive pancreatic orgastric cancer (Ramani et al., 2008; Yashiro et al., 2006). The latter,as well as the finding that JO-1 triggers EMT-like signaling, mightraise the question of whether JO-1 would facilitate metastasis. Notably,in all models used in this study, we did not see stimulation of tumorgrowth or macroscopic/microscopic signs of metastasis in animals treatedwith JO-1 alone. Tumor invasion and metastasis requires more thantransient activation of EMT pathways. Detachment from epithelial cancersand migration of tumor cells is only possible after long-term crosstalkbetween malignant cells and the tumor microenvironment, resulting inchanges in the tumor stroma and phenotypic reprogramming of epithelialcells into mesenchymal cells (Guarino, 2007).

In summary, the epithelial junction opener JO-1 has the potential toimprove mAb therapies of cancer both in term of efficacy and safety,i.e. by allowing lower therapeutic mAbs doses. This study also shedslight on the mechanisms of Ad3 infection of epithelial cells.

Material and Methods

Proteins and antibodies. The generation of JO-1 (also known asAd3-K/S/Kn) has been described previously (Wang et al., 2011a). JO-1 wasproduced in E. coli using the pQE30 expression vector (Qiagen, Valencia,Calif.) and purified by Ni-NTA agarose chromatography as describedelsewhere (Wang et al., 2007). Recombinant Ad3 penton-dodecahedral(PtDd) protein complexes were produced in insect cells and purified asdescribed elsewhere (Fender et al., 1997).

The following antibodies were used for immunofluorescence studies orWestern blot: polyclonal goat anti-DSG2 (R&D Systems, Inc., Minneapolis,Minn.), mouse mAb anti-DSG2 (clone 6D8) (Cell Sciences, Canton, Mass.),rabbit anti-Claudin 7 (Abcam, Cambridge, Mass.), anti-human IgG-FITC(Santa Cruz), rabbit anti-EGFR (Abcam), mouse anti-Her2/neu (Abcam),mouse anti-E-cadherin (Cell Signaling), mouse anti-human IgG Fc (R&DSystems), mouse mAb against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)(Cell Signaling), mouse anti-Erk1/2 (Cell Signaling), mouseanti-phospho-E-cadherin (pS838/840) (Epitomics, Inc.), mouse anti-6-His(Qiagen).

Cell lines. BT474-M1 cells were cultured in DMEM/F12 with 10% FBS, 1%Pen/Strep and 2 mM L-Glutamine. HCC1954 breast cancer, A549 lung cancerand NCI-N87 ???? cells were cultured in RMPI with 10% FBS and 1%Pen/Strep. SKOV3-ip1 cells were cultured in MEBM medium. Colon cancerT84 cells were cultured in a 1:1 mixture of Ham's F12 medium and DMEM,with the addition of 10% FBS, Glu and Pen/Strep. To achieve cellpolarization, 1.4×10⁵ T84 cells were cultured in collagen-coated 6.5 mmTranswell inserts (0.4 μm pore size) (Costar Transwell Clears) for aperiod of 14 to 20 days until transepithelial resistance was stable(Wang et al., 2011b).

Immunofluorescence analyses. Cells were cultured in chamber glass slides(BD Falcon), washed twice with ice-cold PBS and then fixed withmethanol/acetone (1:1 vol/vol) for 15 min at 4° C., or with 4%paraformaldehyde for 30 min at 4° C. Cells were washed twice with PBSafter fixation and subsequently blocked with PBS containing 2% dry-milkpowder (BioRad) for 20 min at room temperature. Antibodies were dilutedin PBS (Claudin7 1:100, DSG2 1:100, Her2/neu 1:50, EGFR 1:50, E-cadherin1:100, Human IgG 1:400, phospho-p44/42 MAPK 1:400, Penta-His 1:500) andcells were stained for 90 min at room temperature or at 4° C. overnight.When required, suitably directed secondary antibodies directed wereapplied after 3 washes with PBS for a period of 30 min at roomtemperature. Glass slides were mounted using VECTASHIELD with DAPI(Vector Labs). Photographs were obtained with a Leica DFC300FX digitalcamera. Confocal images were taken on a Zeiss META Confocal Microscopeusing 40× or 100× oil lenses and Zeiss 510 software (Zeiss MicroImaging,Thornwood, N.Y.).

Tissues. Paraffin sections of human breast cancers were deparaffinizedand rehydrated. Antigen retrieval was performed with Vector AntigenUnmasking solutions pH 6.0 (Vector laboratories, Inc., Burlingame,Calif.). Mouse anti-DGS2 antibody (3G132) (Abcam) was diluted 1:10.Immunohistochemistry was performed using the Polink-2 HRP Broad Kit(Golden Bridge International, Inc. Mukilteo, Wash.) and DAB as asubstrate.

Western Blot. Xenograft tumor tissue was dissected, manually homogenized(tissue disruptor) and incubated for 30 min in protein lysis buffer [20mM Hepes (pH 7.5), 2 mM EGTA, 10% glycine, 1% TritonX100, 150 mM NaCl(all from Sigma-Aldrich), and protease/phospatase inhibitors (CompleteProtease Inhibitor Cocktail and PhosSTOP, Roche)] on ice. Confluentcultured cells were washed twice with ice-cold PBS and then lysed for 30min in protein lysis buffer on ice. Samples were pelleted (10 min, 4°C., 15,000 RPM, and the protein containing supernatant stored at −80° C.A total of 80 μg of total protein was used for the Western blotprocedure. Protein samples were boiled (5 min at 95° C.) and separatedby polyacrylamide gel electrophoresis (PAGE) using 10% Bis-Tris gels andMOPS buffer (Novex, InVitrogen), followed by transfer ontonitrocellulose membranes according to the supplier's protocol (iBlot,InVitrogen). Membranes were blocked in PBS containing 0.1% Tween20(PBS-T, Sigma) and 5% dry milk powder. Incubation times for primary andsecondary antibodies were 16 h at 4° C. and 1 h at room temperature,respectively. Antibodies were diluted [Human IgG 1:1000, DSG2 1:500,Ad3-K serum 1:2000, p-E-cadherin 1:2000, E-cadherin 1:1000, Claudin71:500, Vimentin 1:2000, phospho-p44/42 MAPK (Erk1/2) 1:2000, Erk1/21:2000] in PBS-T and 5% dry-milk powder. Membranes were washed 5 timesin PBS-T between antibody incubations, and films were developed usingECL plus (Amersham) or the Odyssey system (LI-COR Biosciences).

DSG2 mRNA PCR: Reverse transcription was performed on 1 μg of total RNAusing the Quantitect Reverse Transcription Kit (Qiagen) to evaluate theexpression levels of DSG2 in breast cancer. The expression of thehousekeeping gene GAPDH was used as reference. The quantitative PCR(qPCR) was run in triplicates using the SensiMix SYBR Kit (Quantace) ona 7900HT Fast Real-Time PCR System (Applied Biosystems/LifeTechnologies). The following primers were used:

DSG2 QT-PCR fw (SEQ ID NO: 58) 5′-ATG ACG GCT AGG AAC ACC AC-3′DSG2 QT-PCR rev (SEQ ID NO: 59) 5′-TCA GGT ACA TTG GAA ACA TGA AA-3′GAPDH QT-PCR fw (SEQ ID NO: 60) 5′-TGC ACC ACC AAC TGC TTA GC-3′GAPDH QT-PCR rev (SEQ ID NO: 61) 5′-GGC ATG GAC TGT GGT CAT GAG-3′

The qPCR was performed under the following conditions: after an initial10 min enzyme activation step at 95° C., 40 amplification cycles wererun, each consisting of 95° C. for 15 s and 60° C. for 1 min. Lastly, afinal elongation step was performed for two minutes at 60° C.

Permeability assay. A total of 5×10⁵ T84 cells were seeded on 12 mmtranswell inserts [PET membrane, with 0.4 μm pore size (Corning, N. Y.)]and cultured for 14-20 days until transepithelial resistance was stable.Culture medium was changed every 2-3 days. The cells were exposed toDSG2 ligands (20 μg/ml) in adhesion medium (DMEM, 1% FBS, 2 mM MgCl₂, 20mM HEPES) for 15 min at room temperature. Subsequently, 1 mCi of [¹⁴C]polyethylene glycol-4000 (PEG-4000) (Perkin Elmer, Covina Calif.)diluted with DMEM/F12 medium, was added to the inner chamber. Mediumaliquots were harvested from the inner and outer chambers at 15 and 30min and measured by a scintillation counter. Permeability was calculatedas described elsewhere (Yang et al., 2004).

Trastuzumab and cetuximab cytotoxicity assay. BT474 or A549 cells wereplated at a density of 5×10⁴ cells/well in triplicate in 96-well platesand grown to confluence. JO-1 (500 ng/ml) was added to the medium. 12 hlater, trastuzumab or cetuximab (15 μg/ml) were added and cell viabilitywas measured 2 h later by WST-1 assay (Roche, San Francisco, Calif.).Three independent studies were performed.

Electron microscopy. Polarized cells in the transwell chambers werefixed with half-strength Karnovsky's fixative (2% paraformaldehyde, 2.5%glutaraldehyde, and 0.2 M Cacodylate buffer) for 1 h at roomtemperature. The fixative in the inner chamber contained 0.2% rutheniumred. The ruthenium red [Ruthenium(III) chloride oxide, ammoniated], waspurchased from Alfa Aesar (Ward Hill, Mass.). Post-fixation wasperformed with 1% OsO₄-phosphate buffer. The membranes were cut out fromthe transwell chambers and embedded in Medcast (Ted Pella, Redding,Calif.). Ultra-thin sections were stained with uranyl acetate and leadcitrate. Processed grids were evaluated with a JEOL JEM1200EXIItransmission electron microscope. Images were acquired with an OlympusSIS Morada Digital CCD camera using ITEM software for image processing.

Animal studies: All experiments involving animals were conducted inaccordance with the institutional guidelines set forth by the Universityof Washington. Mice were housed in specific-pathogen-free facilities.Breast cancer xenografts were established by injecting 4×10⁶ cancercells into the mammary fat pad of CB17 SCID-beige mice. Trastuzumab wasinjected intraperitoneally (i.p.) at a dose of 10 mg/kg. PtDd or JO-1was given intravenously (i.v.) at a dose of 2 mg/kg. Tumor volumes weremeasured as described previously (Tuve et al., 2007). Mice weresacrificed when the tumor volume reached 1,000 mm³ or ulcerated. Lungcancer xenografts were established by injecting 4×10⁶ A549subcutaneously (s.c.) into the right flank of CB17 SCID beige mice.Cetuximab was injected at 10 mg/kg i.p. 10 h after JO-1 i.v. injectionat day 12 and repeated after 3 days. For the disseminated lung tumormodel, eight week old male CB17 SCID-beige mice were intravenouslyinjected with 2×10⁶ A549 cells on day 1. The treatment with JO-1 (2mg/kg i.v) and cetuximab (10 mg/kg i.p.) was started at day 10 andrepeated every three days. Animals were monitored for weight loss andsigns of dyspnea. Animals were sacrificed when the first mouse of thenon-treatment group was moribund. India ink (15% in PBS) was injectedintratracheally prior to the removal of the lungs. Metastases appearedunstained (white) against the black normal tissue. To produce anti-JO-1antibodies, mice were injected subdermally with JO-1 mixed with Freund'sadjuvant as described elsewhere (Li et al., 2009).

HSC-based relaxin expression: The protocol has been described elsewhere(Beyer et al., 2011). Briefly, transplant recipients were 6- to 10-weekold, female CB17 SCID-beige mice, sublethally irradiated with 350 cGyimmediately before tail vein injection with 6×10⁶ lentivirusvector-transduced bone marrow cells from 5-FU-treated mice. Afterengraftment of cells in the recipients' bone marrow was confirmed, atotal of 4×10⁶ HCC1954 were injected into the mammary fat pad. Thelentivirus vector expressing relaxin under the control of doxycycline(Dox) has been described previously (Beyer et al., 2011).

Neutralizing antibody assay: Serum samples from cancer patients weretaken before patients underwent chemotherapy. Briefly, 293 cells wereplated in 96-well plates at 4×10⁴ cells per well and incubated at 37° C.The following day, serum samples were heat inactivated at 56° C. for 30min and serially diluted from 1:2 to 1:1, 204 in MEM containing 2% FCS.A total of 20 plaque forming units (PFU) per cell of wild-type Ad5 orAd11 in 10 μl of MEM, was incubated with 100 μl of each serum dilutionfor 1 h at 37° C. Medium was removed from cells plated the previous day,and 55 μl of virus-containing serum was added to the cells along with 45μl of 293 growth medium. A further 100 μl of 293 growth medium was addedto the cells at 3 and 6 days postinfection. At 8 days postinfection,cells were analyzed for the presence of cytopathic effect (CPE), andserum samples were scored positive for the presence of neutralizingantibodies if no CPE was seen at a dilution of 1:2 or higher.

Statistical analysis: All results are expressed as mean 41-SD. Student'st-test or 2-Way ANOVA for multiple testing, were applied whenapplicable. A p-value<0.05 was considered significant.

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1. A recombinant AdB-2/3 fiber polypeptide, comprising: a) one or moreAdB-2/3 fiber polypeptide shaft domains; b) an AdB-2/3 fiber polypeptideknob domain, operatively linked to and located C-terminal to the one ormore AdB-2/3 fiber polypeptide shaft domains; and c) one or morenon-AdB-2/3-derived dimerization domains operatively linked to andlocated N-terminal to the one or more AdB-2/3 fiber polypeptide shaftdomains.
 2. The recombinant AdB-2/3 fiber polypeptide of claim 1,wherein the AdB-2/3 fiber polypeptide does not include an AdB-2/3 fiberpolypeptide tail domain.
 3. The recombinant AdB-2/3 fiber polypeptide ofclaim 1, wherein each shaft domain is selected from the group consistingof an Ad3 fiber polypeptide shaft domain, an Ad7 fiber polypeptide shaftdomain, an Ad11 fiber polypeptide shaft domain, an Ad 14 fiberpolypeptide shaft domain, an Ad14a fiber polypeptide shaft domain, andcombinations thereof.
 4. The recombinant AdB-2/3 fiber polypeptide ofclaim 1, wherein the one or more shaft domains comprise 1-22 shaftdomains.
 5. The recombinant AdB-2/3 fiber polypeptide of claim 1,wherein each shaft domain comprises an amino acid sequence according toSEQ ID NO 11: GVL(T/S)LKC(L/V)(T/N)PLTT(T/A)(G/S)GSLQLKVG(G/S)GLTVD(D/T)T(D/N)G(T/F/S)L(Q/K/E)ENI(G/S/K)(A/V)(T/N)TPL(V/T)K(T/S)(G/N)HSI(G/N)L(S/P)(L/I)G(A/P/N)GL(G/Q)(T/I)(D/E)(E/Q)NKLC(T/S/A)KLG(E/Q/N)GLTF(N/D)S(N/S)N(I/S)(C/I)(I/A)(D/N/L)(D/K)N(I/--)NTL.


6. The recombinant AdB-2/3 fiber polypeptide of claim 1, wherein eachshaft domain comprises an amino acid sequence according to SEQ ID NO 12:GVLTLKCLTPLTTTGGSLQLKVGGGLT(V/I)DDTDG(T/F)L(Q/K)ENI(G/S)ATTPLVKTGHSIGL(S/P)LG(A/P)GLGT(D/N)ENKLC(T/A)KLG(E/Q)GLTFNSNNICI(D/N)DNINTL.


7. The recombinant AdB-2/3 fiber polypeptide of any claim 1, whereineach shaft domain comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEID NO:4,and SEQ ID NO:5.
 8. The recombinant AdB-2/3 fiber polypeptide of claim1, wherein the knob domain is selected from the group consisting of anAd3 fiber polypeptide knob domain, an Ad7 fiber polypeptide knob domain,an Ad11 fiber polypeptide knob domain, an Ad 14 fiber polypeptide knobdomain, an Ad14a fiber polypeptide knob domain.
 9. The recombinantAdB-2/3 fiber polypeptide of claim 1, wherein the knob domain comprisesan amino acid sequence according to SEQ ID NO 13:WTG(V/P)(N/K)P(T/)(E/R)ANC(Q/I)(M/I)(M/E)(Y/A/N/D)(S/K)(S/K)(E/Q)(S/N)(N/P)D(C/S)KL(I/T)L(I/T)LVK(T/N)G(A/I)(L/I)V(T/N)(A/G)(F/Y)V(Y/T)(V/L)(I/M)G(V/A)S(N/D)(N/D/Y)(F/V)N(M/T)L(T/F)(T/K)(Y/H/N)(R/K)N(I/V)(N/S)(F/I)(T/N)(A/V)EL(F/Y)FD(S/A)(A/T)G(N/H)(L/I)L(T/P)(S/R/D)(L/S)SSLKT(P/D)L(N/E)(H/L)K(S/Y)(G/K)Q(N/T)(M/--)(A/--)(T/--)(G/--)A(I/L/D)(T/F)(N/S)A(K/R)(S/G)FMPSTTAYPF(--/V)(--/L)(N/P)(N/D/V)(N/A)(S/G)(R/T)(E/H)(N/K/--)(--/E)NYI(Y/F)G(T/Q)C(H/Y)Y(T/K)ASD(H/G)(T/A)(A/L)FP(I/L)(D/E)(I/V)(S/T)VMLN(Q/R/K)R(A/L)(I/L/P)(R/N/D)(A/D/N/S)(D/E/R)TSY(C/V)(I/M)(R/T)(I/V/F)(T/L)WS(W/L)N(T/A)G(D/L/V)APE(G/V/--)(Q/--)T(S/T)(A/Q)(T/A)TL(V/I)TSPFTF(Y/S)YIREDD.


10. The recombinant AdB-2/3 fiber polypeptide of claim 1, wherein theknob domain comprises an amino acid sequence according to SEQ ID NO 14:WTGVNPT(E/R)ANCQ(M/I)(M/I)(D/N/A)SSESNDCKLILTLVKTGALVTAFVYVIGVSN(N/D)FNMLTT(Y/H)(R/K)NINFTAELFFDS(A/T)GNLLT(S/R)LSSLKTPLNHKSGQNMATGA(I/L)TNAK(S/G)FMPSTTAYPFN(N/D/V)NSRE(--/K)(-/E)NYIYGTC(H/Y)YTASD(H/R)TAFPIDISVMLN(Q/R)RA(I/L)(R/N)(A/D/N)(D/E)TSYCIR(I/V)TWSWNTG(D/V)APE(G/V)QTSATTLVTSPFTFYYIREDD.


11. The recombinant AdB-2/3 fiber polypeptide of claim 1, wherein theknob domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEID NO:9, and SEQID NO:10.
 12. The recombinant AdB-2/3 fiber polypeptide of claim 1,wherein the dimerization domain comprises an amino acid sequenceselected from the group consisting of EVSALEK (SEQ ID NO:22) and/orKVSALKE (SEQ ID NO: 23).
 13. The recombinant AdB-2/3 fiber polypeptideof claim 1, wherein the recombinant AdB-2/3 polypeptide comprises: a)one or more shaft domains that each comprise an Ad3 shaft domain (SEQ IDNO:1); and b) a knob domain that comprises an Ad3 knob domain (SEQ IDNO:6).
 14. The recombinant AdB-2/3 fiber polypeptide of claim 13,comprising the amino acid sequence of junction opener-1 (JO-1) (SEQ IDNO:20).
 15. The recombinant AdB-2/3 fiber polypeptide of claim 1,wherein the AdB-2/3 fiber polypeptide contains a single AdB-2/3 fiberpolypeptide shaft domain.
 16. The recombinant AdB-2/3 fiber polypeptideof claim 1, wherein the AdB-2/3 fiber polypeptide is multimerized. 17.The recombinant AdB-2/3 fiber polypeptide of claim 1, wherein theAdB-2/3 fiber polypeptide is dimerized.
 18. The recombinant AdB-2/3fiber polypeptide of claim 1, further comprising one or more compoundsconjugated to the recombinant AdB-2/3 fiber polypeptide.
 19. Therecombinant AdB-2/3 fiber polypeptide of claim 18, wherein the one ormore compounds are selected from the group consisting of therapeutics,diagnostics, and imaging agents.
 20. The recombinant AdB-2/3 fiberpolypeptide of claim 19, wherein the one or more compounds comprise atleast one therapeutic, wherein the therapeutic is selected from thegroup consisting of antibodies, immunoconjugates, nanoparticles,chemotherapeutics, radioactive particles, viruses, vaccines, cellularimmunotherapy therapeutics, gene therapy constructs, nucleic acidtherapeutics, and combinations thereof.
 21. An isolated nucleic acidencoding the recombinant AdB-2/3 fiber polypeptide of claim
 1. 22. Arecombinant expression vector comprising the isolated nucleic acid ofclaim
 21. 23. A host cell comprising the recombinant expression vectorof claim
 22. 24. A pharmaceutical composition, comprising a) an AdB-2/3fiber multimer; and b) a pharmaceutically acceptable carrier.
 25. Thepharmaceutical composition of claim 24, wherein the AdB-2/3 fibermultimer comprises the multimerized recombinant AdB-2/3 fiberpolypeptide of any one of claims 16-20.
 26. A method for enhancingtherapeutic treatment, or diagnosis of a disorder associated withepithelial tissue, and/or imaging epithelial tissues, comprisingadministering to a subject in need thereof: a) an amount of one or moretherapeutics sufficient to treat the disorder, diagnostic sufficient todiagnose the disorder, and/or imaging agent sufficient to image theepithelial tissue; and b) an amount of AdB-2/3 fiber multimer sufficientto enhance efficacy of the one or more therapeutics, diagnostics, and/orimaging agents.
 27. The method of claim 26, wherein the disorderassociated with epithelial tissue is selected from the group consistingof solid tumors, irritable bowel syndrome, inflammatory bowel disorder,Crohn's disease, ulcerative colitis, constipation, gatroesophagealreflux disease, Barrett's esophagus, chronic obstructive pulmonarydisease, asthma, bronchitis, pulmonary emphysema, cystic fibrosis,interstitial lung disease, pneumonia, primary pulmonary hypertension,pulmonary embolism, pulmonary sarcoidosis, tuberculosis, pancreatitis,pancreatic duct disorders, bile duct obstruction, cholecystitis,choledocholithiasis, brain disorders, psoriasis, dermatitis,glomerulonephritis, hepatitis, diabetes, thyroid disorders, cellulitis,infection, pyelonephritis, and gallstones.
 28. The method of claim 26,wherein the disorder associated with epithelial tissue is a solid tumor.29. The method of claim 28 wherein the solid tumor is selected from thegroup consisting of breast tumors, lung tumors, colon tumors, rectaltumors, stomach tumors, prostate tumors, ovarian tumors, uterine tumors,skin tumors, endocrine tumors, cervical tumors, kidney tumors,melanomas, pancreatic tumors, liver tumors, brain tumors, head and necktumors, nasopharyngeal tumors, gastric tumors, squamous cell carcinomas,adenocarcinomas, bladder tumors, and esophageal tumors.
 30. The methodof claim 26, wherein the AdB-2/3 fiber multimer is selected from thegroup consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, anAd11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer,and combinations thereof.
 31. The method of claim 26 wherein the AdB-2/3fiber multimer is an Ad3 fiber multimer.
 32. The method of claim 26,wherein the AdB-2/3 fiber multimer is selected from the group consistingof AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles(PtDd), and recombinant AdB-2/3 fiber multimers.
 33. The method of claim26, wherein the AdB-2/3 fiber multimer comprises an Ad3 PtDd.
 34. Themethod of claim 26, wherein the AdB-2/3 fiber multimer comprisesjunction opener 1 (JO-1) (SEQ ID NO:20).
 35. The method of claim 26wherein one or more compounds comprises at least one therapeutic,wherein the therapeutic is selected from the group consisting ofantibodies, immunoconjugates, viruses, nanoparticles, chemotherapeutics,radioactive particle, vaccines, cellular immunotherapy therapeutics,gene therapy constructs, nucleic acid therapeutics, and combinationsthereof.
 36. The method of claim 26, wherein the therapeutic comprises achemotherapeutic or a monoclonal antibody.
 37. The method of claim 26,wherein the therapeutic comprises an anti-tumor monoclonal antibody. 38.The method of claim 37, wherein the anti-tumor monoclonal antibodycomprises an antibody selected from the group consisting of trastuzumab,cetumiximab, petuzumab, apomab, conatumumab, lexatumumab, bevacizumab,bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab,ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab,gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab,catumaxomab, ofatumumab, and ibritumomab.
 39. The method of claim 26,wherein the disorder associated with epithelial tissue comprises a Her-2positive tumor.
 40. The method of claim 39, wherein the Her-2 positivetumor is selected from the group consisting of a breast tumor, a gastrictumor, a colon tumor, and an ovarian tumor.
 41. The method of claim 39,wherein the therapeutic comprises trastuzumab.
 42. The method of claim39 wherein the therapeutic comprises a chemotherapeutic, radiation, orcombinations thereof.
 43. The method of claim 39, wherein the AdB-2/3fiber multimer comprises an Ad3 PtDd, or JO-1.
 44. The method of claim39, wherein the subject has not responded to trastuzumab therapy. 45.The method of claim 26, wherein the disorder associated with epithelialtissue comprises an EGFR-positive tumor.
 46. The method of claim 45,wherein the EGFR-positive tumor is selected from the group consisting ofa lung tumor, a colon tumor, a breast tumor, a rectal tumor, a head andneck tumor, and a pancreatic tumor.
 47. The method of claim 45, whereinthe therapeutic comprises cetuximab.
 48. The method of claim 28, whereinthe therapeutic comprises a VEGF inhibitor.
 49. The method of claim 45wherein the therapeutic comprises a chemotherapeutic, radiation, orcombinations thereof.
 50. The method of claim 45 wherein the AdB-2/3fiber multimer comprises an Ad3 PtDd, JO-1.
 51. The method of claim 45,wherein the subject has not responded to cetuximab therapy.
 52. Themethod of claim 26, wherein the AdB-2/3 fiber multimer is a dimer. 53.The method of claim 52, wherein the AdB-2/3 fiber multimer comprisesJO-1.
 54. A method for treating a disorder associated with epithelialtissue, comprising administering to a subject in need thereof an amountof AdB-2/3 fiber multimer sufficient to treat the disorder.
 55. Themethod of claim 54, wherein the disorder is an AdB-2/3 viral infectionor a solid tumor.
 56. The method of claim 55, wherein the disorder is asolid tumor, wherein the solid tumor is selected from the groupconsisting of breast tumors, lung tumors, colon tumors, rectal tumors,stomach tumors, prostate tumors, ovarian tumors, uterine tumors, skintumors, endocrine tumors, cervical tumors, kidney tumors, melanomas,pancreatic tumors, liver tumors, brain tumors, head and neck tumors,nasopharyngeal tumors, gastric tumors, squamous cell carcinomas,adenocarcinomas, bladder tumors, and esophageal tumors.
 57. The methodof claim 54, wherein the AdB-2/3 fiber multimer is selected from thegroup consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, anAd11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer,and combinations thereof.
 58. The method of claim 54 wherein the AdB-2/3fiber multimer is an Ad3 fiber multimer.
 59. The method of claim 54,wherein the AdB-2/3 fiber multimer is selected from the group consistingof AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles(PtDd), recombinant AdB-2/3 fiber multimers.
 60. The method of claim 54,wherein the AdB-2/3 fiber multimer comprises an Ad3 PtDd.
 61. The methodof claim 54, wherein the AdB-2/3 fiber multimer comprises junctionopener 1 (JO-1) (SEQ ID NO:20).
 62. The method of claim 54, wherein theAdB-2/3 fiber multimer is a dimer.
 63. The method of claim 62, whereinthe AdB-2/3 fiber multimer comprises JO-1.
 64. A method for improvingdelivery of a compound to an epithelial tissue, comprising contactingthe epithelial tissue with a) one or more compounds to be delivered tothe epithelial tissue; and b) an amount of AdB-2/3 fiber multimersufficient to enhance delivery of the one or more compounds to theepithelial tissue.
 65. The method of claim 64 wherein the one or morecompounds comprises a diagnostic or an imaging agent.
 66. The method ofclaim 65, wherein the epithelial tissue comprises a solid tumor.
 67. Themethod of claim 66, wherein the solid tumor is selected from the groupconsisting of breast tumors, lung tumors, colon tumors, rectal tumors,stomach tumors, prostate tumors, ovarian tumors, uterine tumors, skintumors, endocrine tumors, cervical tumors, kidney tumors, melanomas,pancreatic tumors, liver tumors, brain tumors, head and neck tumors,nasopharyngeal tumors, gastric tumors, squamous cell carcinomas,adenocarcinomas, bladder tumors, and esophageal tumors.
 68. A method forimproving delivery of a substance to a tissue expressing desmoglein 2(DSG2), comprising contacting the tissue expressing DSG2 with a) one ormore compound to be delivered to the tissue; and b) an amount of AdB-2/3fiber multimer sufficient to enhance delivery of the one or morecompounds to the tissue.
 69. A method for inducing an epithelial tomesenchymal transition (EMT) in a tissue, comprising contacting theepithelial tissue with an amount of AdB-2/3 fiber multimer sufficient toinduce EMT.
 70. The method of claim 64, wherein the AdB-2/3 fibermultimer is selected from the group consisting of an Ad3 fiber multimer,an Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer,an Ad14a fiber multimer, combinations thereof.
 71. The method of claim64 wherein the AdB-2/3 fiber multimer is an Ad3 fiber multimer.
 72. Themethod of claim 64, wherein the AdB-2/3 fiber multimer is selected fromthe group consisting of AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3dodecahedral particles (PtDd), recombinant AdB-2/3 fiber multimers. 73.The method of claim 64 wherein the AdB-2/3 fiber multimer comprises anAd3 PtDd.
 74. The method of claim 64, wherein the AdB-2/3 fiber multimercomprises junction opener 1 (JO-1) (SEQ ID NO:20).
 75. The method ofclaim 64, wherein the AdB-2/3 fiber multimer is a dimer.
 76. The methodof claim 75, wherein the AdB-2/3 fiber multimer comprises JO-1 (SEQ IDNO:20).
 77. A method for identifying candidate compounds for one or moreof treating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, for improving deliveryof a substance tissue expressing DSG2, inducing an EMT in a tissue,and/or treating an AdB-2/3 infection comprising a) contacting an AdB-2/3fiber multimer to DSG2 under conditions to promote multimer binding toDSG2, wherein the contacting is carried out in the presence of one ormore test compounds; and b) identifying positive test compounds thatcompete with the AdB-2/3 fiber multimer for binding to DSG2 compared tocontrol; wherein the positive test compounds are candidate compounds forone or more of treating a disorder associated with epithelial tissue,improving delivery of a substance to an epithelial tissue, for improvingdelivery of a substance tissue expressing DSG2, inducing an EMT in atissue, and/or treating an AdB-2/3 infection.
 78. The method of claim77, wherein the AdB-2/3 fiber multimer is selected from the groupconsisting of an Ad3 fiber multimer, an Ad7 fiber multimer, an Ad11fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer,combinations thereof.
 79. The method of claim 77 wherein the AdB-2/3fiber multimer is an Ad3 fiber multimer.
 80. The method of claim 77,wherein the AdB-2/3 fiber multimer is selected from the group consistingof AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles(PtDd), recombinant AdB-2/3 fiber multimers.
 81. The method of claim 77,wherein the AdB-2/3 fiber multimer comprises an Ad3 PtDd.
 82. The methodof any one of claims 77-80, wherein the AdB-2/3 fiber multimer comprisesjunction opener 1 (JO-1) (SEQ ID NO:20).
 83. The method of claim 77,wherein the AdB-2/3 fiber multimer is a dimer.
 84. The method of claim83, wherein the AdB-2/3 fiber multimer comprises JO-1.
 85. Thepharmaceutical composition of claim 24, wherein the AdB-2/3 fibermultimer comprises a JO-1 dimer.
 86. The pharmaceutical composition ofclaim 24, further comprising an amount of one or more therapeuticssufficient to treat a disorder associated with epithelial tissue, adiagnostic sufficient to diagnose a disorder associated with epithelialtissue, and/or an imaging agent sufficient to image an epithelialtissue.